Use of cd34+ hematopoietic progenitor cells for the treatment of cns disorders

ABSTRACT

The present invention provides novel methods for delivering cells, particularly modified cells to the central nervous system (CNS). The purpose of this invention is to present a method that provides sustained delivery of a molecule to the central nervous system, thereby increasing the bioavailability of the molecule and lengthening the possible duration of treatment.

FIELD OF THE INVENTION

The inventions relates to cell therapy, particularly the use of cellcompositions enriched in hematopoietic progenitor cells to delivertherapeutic molecules to the central nervous system of a mammal,particularly of a human.

BACKGROUND

Many diseases of the CNS in general lack effective treatment due to lackof adequate mechanism for the delivery of therapeutic molecules. Variousdrug delivery systems have been designed by using carriers such asproteins, peptides, polysaccharides, synthetic polymers, colloidalparticles (i.e., liposomes, vesicles or micelles), microemulsions,microspheres and nanoparticles. These carriers, which contain entrappedpharmaceutically useful agents, are intended to achieve controlledcell-specific or tissue-specific drug release. Further efforts andresearch are being directed to develop and design novel systems ofspecific delivery to a target cell or tissue for the agents that crossbiological barriers at relatively low rates.

In order to exert desired therapeutic or prophylactic effects,therapeutic molecules must reach brain cells and tissue. Intravenousadministration will require their passage from the blood to the brain bycrossing the microcapillary membranes of the cerebrovascular endotheliumalso called the blood-brain barrier. Briefly, the blood-brain barrier(BBB) is formed by a monolayer of tightly connected microvascularendothelial cells with anionic charges. This layer separates twofluid-containing compartments: the blood plasma (BP) and extracellularfluid (ECF) of the brain parenchyma, and is surrounded by astroglialcells of the brain. One of the main functions of the BBB is to regulatethe transfer of components between the BP and the ECF. The BBB limitsfree passage of most agent molecules from the blood to the brain cells.

In general, large molecules of high polarity, such as peptides,proteins, (e.g. enzymes, growth factors and their conjugates,oligonucleotides, genetic vectors and others) do not cross the BBB.Therefore poor agent delivery to the CNS limits the applicability ofsuch macromolecules for the treatment of neurodegenerative disorders andneurological diseases.

Several delivery approaches of therapeutic molecules to the braincircumvent the BBB. Such approaches utilize intrathecal injections,surgical implants and interstitial infusion. These strategies deliver anagent to the CNS by direct administration into the cerebrospinal fluid(CSF) or into the brain parenchyma (ECF).

Diffusion of macromolecules to various areas of the brain byconvection-enhanced delivery is another method of administrationcircumventing the BBB. This method consists of: a) creating a pressuregradient during interstitial infusion into white matter to generateincreased flow through the brain interstitium (convection-supplementingsimple diffusion); b) maintaining the pressure gradient over a lengthyperiod of time (24 hours to 48 hours) to allow radial penetration of themigrating compounds (such as: neurotrophic factors, antibodies, growthfactors, genetic vectors, enzymes, etc.) into the gray matter; and c)increasing drug concentrations by orders of magnitude over systemiclevels.

In an attempt to provide a constitutive supply of drugs or other factorsto the brain or other organs or tissues at a controlled rate, miniatureosmotic pumps have been used. However, limited solubility and stabilityof certain drugs, as well as reservoir limitations, have restricted theusefulness of this technology. For example, controlled sustained releaseof dopa mine has been attempted by implanting dopamine encapsulatedcells within bioresorbable microcapsules (McRae-Degueurce et al., 1988,Neurosci. Lett 92:303-309). However, controlled sustained release of adrug from a bioresorbable polymer may rely, e.g., on bulk or surfaceerosion, which may be due to various hydrolytic events. Erosion oftenrelies on hydrolytic events which increase the likelihood of drugdegradation, and complicates establishment of predictable release rates.Other disadvantages associated with pumps and resorbable polymersinclude finite loading capabilities and the lack of feedback regulation.

Another strategy to improve agent delivery to the CNS is by increasingthe molecules' absorption (adsorption and transport) through the BBB andtheir uptake by the cells [Broadwell, Acta Neuropathol., 79:117-128,1989; Pardridge et al., J. Phaimacol. Experim. Therapeutics,255(2):893-899, 1990; Banks et al., Progress in Brain Research,91:139-148, 1992; Pardridge, Fuel Homeostasis and the Nervous System,Edited by Vranic et al., Plenum Press, New York, 43-53, 1991]. Thepassage of agents through the BBB to the brain can be enhanced byimproving either the permeability of the agent itself or by altering thecharacteristics of the BBB. Thus, the passage of the agent can befacilitated by increasing its lipid solubility through chemicalmodification, and/or by its coupling to a cationic carrier, or still byits covalent coupling to a peptide vector capable of transporting theagent through the BBB. Peptide transport vectors are also known as BBBpermeabilizer compounds.

In other examples, direct administration to the CNS has been used todelivery molecules that would otherwise not pass the blood brainbarrier. For example, polypeptides as well as viral vectors capable ofdirecting the expression of a therapeutic polypeptide have beendelivered intracerebrally and intrathecally. However, directadministration for the delivery of polypeptides has the evidentconvenience disadvantages due to repeated administration, and directadministration for the delivery of nucleic acids using viral vectors notbeen capable of achieving widespread transduction of cells beyond thesite of administration.

Thus, the disadvantages of all of these approaches present a significantobstacle to the development of therapies of the treatment of CNSdisorders, particularly those that involve a widespread population ofneurons or glial cells.

Cell Transplantation

Cell transplantation, including ex vivo gene therapy has also beenpursued as a therapeutic strategy, as for example in Bachoud-levi A C etal., (Lancet 2000, 356:1975-79) for Huntington's disease. However, thedifficulties encountered vary greatly depending on the application andthe cell source considerations, as exemplified in Table 1 (reproducedfrom Gage et al., Nature 392 (supp):18-24, 1998) below. TABLE 1 Cellsource Advantages Disadvantages Solution Autologous ImmunologicallyLimited supply; time Cryopreserve; privileged; no ethical constraintsfor donor and multiply in vitro issues host Allogeneic Greater supply;few time Cellular and humoral Immunosuppress; constraints on donorimmunity; encapsulate ethical issues: fetal tissues Xenograft Greatersupply; no time Cellular and humoral Immunosuppress; constraints ondonor immunity; possible encapsulate; transfer of new virus geneticallymask accross species immunity Cell line Infinite supply; no timeCellular and humoral Immunosuppress; (immortalized contraints for donoror tumorigenicity and encapsulate; or tumorigenic) host; safety test andneoplasia genetically mask standardization simplified immunity

Nevertheless, in view of the above-mentioned difficulties with othersystems, alternative treatments for neurodegenerative diseases haveemerged. As a general approach, cells have been transplanted into thearea of neurodegeneration in an effort to reconstitute damaged neuralcircuits, and to replace lost neurons and neurotransmitter systems. Suchtreatments include transplantation of genetically engineered cells (seee.g., Breakefield, X. O. et al., 1989, Neurobiol. Aging 10:647-648;Gage, F. H. et al., 1987, Neuroscience 23:795-807; Horellou P. et al.,1990, Eur. J. Neurosci. 2:116-119; Rosenberg, M. B. et al., 1988,Science 242:1575-1578; Wolff, J. A. et al., 1989, Proc. Natl. Acad. Sci.USA 86:9011-9014) or fetal cells (see e.g., Bjorklund, A. et al., 1983,Acta. Physiol. Scand Suppl. 522:1-75; Dunnett, S. B. et al., 1990, inBrain Repair (eds. Bjorklund, A. et al.) Wenner-Gren InteriationalSymposium Series 56:335-373 (McMillan Press, London); Isacson, O. etal., 1984, Nature 311;458-460; using porcine fibroblasts in U.S. Pat.No. 6,204,053.

In one strategy, engineered cells have been derived from cell lines orgrown from recipient host fibroblasts or other cells and then modifiedto produce and secrete substances following transplantation into aspecific site in the brain. For example, one group of researchersdeveloped a biological system in which genetically engineered nervegrowth factor-producing rat fibroblasts, when implanted into the ratstriatum prior to infusion of neurotoxins were reported to protectneurons from excitotoxin-induced lesions (Schumacher, J. M. et al.,1991, Neuroscience 45(3):561-570). Another group which transplanted ratfibroblasts genetically modified to produce L-DOPA or dopamine into6-hydroxydopamine lesions of the nigrostriatal pathway in rats reportedthat the transplanted fibroblasts reduced behavioral abnormalities inthe lesioned rats (Wolff, J. A. et al., 1989, Proc. Natl. Acad. Sci. USA86:9011-9014). As an alternative to genetically engineered cells, cellsto be implanted into the brain can be selected because of theirintrinsic-release of critical compounds, e.g., catecholamines by PC 12cells and nerve growth factor by immortalized hippocampal neurons.

In other strategies, intracerebral neural grafting has emerged as apotential approach to CNS therapy. The replacement or addition of cellsto the CNS which are able to produce and secrete therapeutically usefulmetabolites may offer the advantage of averting repeated drugadministration while also avoiding the drug delivery complications posedby the blood-brain barrier (Rosenstein, Science 235:772-774, 1987).However, optimization of the survival of grafted cells has proveddifficult, and no convenient and plentiful source of neurons isavailable.

Bone Marrow Transplantation

In yet other strategies, bone marrow transplantation (BMT) has been usedto treat several genetic disorders that affect the CNS. The first groupincludes various lysosomal storage disorders with CNS involvement. Inthese disorders, deficiency of lysosomal enzyme affects primarilyneurons (as in mucopolysaccharodisosis) or oligodendrocytes (as inmetacromatic leukodystrophy or Krabbe disease). The rationale for BMT inthe treatment of these disorders was that monocyte-derived cells fromthe donor can enter the brain, differentiate into microglia and/orperivascular macrophages and secrete normal lysosomal enzymes that canbe recaptured by neurons or oligodendrocytes.

Similar reasoning was used initially by Moser H W et al., Neurology34:1410-1417, 1984, to propose BMT in X-linked adrenoleukodystrophy(ALD). However, the protein to be delivered in X-linkedadrenoleukodystrophy, the ALD protein, was a non-secreted protein, suchthat after the allogenic BMT, only microglia/perivascular macrophagesexpressed the normal ALD protein. The protein thus could not be secretedand recaptured by neurons and other glial cells. BMT for the treatmentof ALD therefore likely represents a true form of “brain cell therapy”.Total BMT, referring to the transplantation of bone marrow cells withouta purification or enrichment step, has also been demonstrated useful inthe treatment of CNS disorders in multiple sclerosis (Burt et al,Inmunol. Today 1997, 18(12):559-561), autoimmune encephalomyelitis (vanGelder et al., Transplantation, 1996, 62(6):810-818, metachromaticleukodystrophy (Matzner et al. 2000, Gene Ther. 7(14):1250-1257), Fabrydisease (Takenaka et al., 2000, PNAS USA 97(13):7515-7520), andgangliosidoses (Norflus et al., 1998, J. Clin. Invest. 101(9):1881-1888;and Oya et al., 2000, Acta Neuropathol. 99(2):161-168).

Transplanting total bone marrow presents several important disadvantages(Gage et al., 1998). Transplantation of whole bone marrow requires thatseveral punctures in bone be made under anesthesia to obtain enoughcells for transplantation. Furthermore, despite evidence suggestingthat 1) monocytes can enter the brain and differentiate intoperivascular macrophages and; 2) cells derived from the donor and havingthe morphological and histochemical characteristic of microglia can berecovered in the recipient mice after bone marrow transplantation, onedoes not know at which stage of differentiation (from early primitiveHSC to already differentiated monocytic stage), hematopoietic cellsenter the brain after bone marrow transplantation and differentiate intomicroglia.

Therefore, there is a need in the art for methods of delivering cells,nucleic acids and/or polypeptides to the CNS. There is accordingly alsoa need for methods of providing a convenient source of cells,particularly modified cells expressing a polypeptide of interest,capable of migrating to the CNS and providing or performing a desiredbiological function over a long period of time (e.g. months or years).

SUMMARY OF THE INVENTION

The present invention provides novel methods for delivering cells,particularly modified cells, to the central nervous system (CNS). Thepurpose of this invention is to present a method that provides sustaineddelivery of a molecule to the central nervous system, thereby increasingthe bioavailability of the molecule and lengthening the possibleduration of treatment.

The invention involves providing a population of cells enriched inhematopoietic stem or progenitor or stem cells capable of migrating tothe CNS upon administration to a subject at a site outside of the CNS.In preferred embodiments, the present invention provides a population ofcells capable of differentiation into cells of the CNS, particularlymicroglia cells. Based on the characterization of populations ofhematopoietic cells capable of giving rise to brain microglia expressinga desired polypeptide, the invention provides hematopoietic progenitoror stem cells and ex vivo therapies to provide cells that migrate to theCNS and differentiate into cell types found in the CNS. Moreover, inpreferred embodiments, the populations of cells include hematopoieticprogenitor or stem cells displaying the CD34 marker, allowing the cellsto be conveniently separated using widely available equipment.

The invention is based on the inventors' demonstration that ex vivogenetic manipulation can be performed wherein human CD34+ cells andderived myelomonocytic cells obtained from ALD patients and transducedwith a HIV-1 derived vector carrying the ALD cDNA can enter into thebrain, differentiate into microglia and express a “therapeutic” protein.In a model of xenograft transplantation, it was demonstrated thatmyelomonocytic cells derived from human CD34+ cells can: 1) enter intothe brain; 2) differentiate into microglia; 3) and express a“therapeutic” protein for several months, once these cells have beengenetically modified ex vivo prior to transplantation.

In one aspect, the invention provides a method of administering anucleic acid or protein of interest to the central nervous system of amammal, comprising providing a composition enriched in hematopoieticprogenitor cells or stem cells, and administering said composition to amammal. Advantageously, at least of portion of said cells furthercomprise a nucleic acid of interest. The method provides particularadvantages for the treatment of a mammal affected by or susceptible tobeing affected by a CNS disorder.

Disclosed is a method of delivering a nucleic acid sequence encoding apolypeptide of interest to a mammal, said method comprising: a)providing a composition enriched in hematopoietic progenitor cells orstem cells, preferably cells expressing the CD34 marker or cells capableof giving rise to cells expressing the CD34 marker, wherein at least aportion of said cells are recombinant cells comprising a nucleotidesequence encoding said polypeptide operably linked to expression controlelements; and b) administering said composition to a mammal underconditions that result in the expression of the polypeptide of interestat a level that provides a therapeutic effect in said mammal.Furthermore, provided is a method of delivering a nucleic acid sequenceencoding a polypeptide of interest to a mammal, said method comprising:a) obtaining cells from a human subject, said cells comprisinghematopoietic progenitor cells or stem cells; b) isolating ahematopoietic progenitor or stem cell from said cells obtained from saidsubject; c) introducing a nucleic acid encoding a polypeptide ofinterest to said hematopoietic progenitor or stem cell; and d)administering said composition to a human subject affected by orsusceptible to being affected by a CNS disorder under conditions thatresult in the expression of a polypeptide of interest at a level thatprovides a therapeutic effect in said mammal.

Encompassed also is a method for delivering a cell, preferably to theCNS of a mammal comprising: a) providing a composition enriched inhematopoietic progenitor cells or stem cells, said cells preferablyexpressing the CD34 marker or capable giving rise to cells expressingthe CD34 marker; and b) administering said composition to a mammal. Theadministered cells will give rise to microglia cells in the CNS.

In preferred aspects of the methods of the invention, the at least ofportion of said cells comprise a nucleic acid of interest. The nucleicacid of interest may encode a secreted or a nonsecreted protein. Thecells of the invention are preferably transduced with a vectorcomprising a nucleic acid of interest operably linked to a promotorcapable of effecting the, expression of said nucleic acid of interest ina hematopoietic cell. The vector is preferably a viral vector, mostpreferably a lentiviral vector.

In preferred aspects of the invention, at least a portion of theadministered hematopoietic progenitor or stem cells are capable ofmigrating to the CNS and/or are capable of expressing the nucleic acidof interest in the CNS, and/or are capable of giving rise to cells ofthe CNS, preferably microglia.

Preferably human hematopoietic progenitor or hematopoietic stem cellsare used in the present invention, most preferably human cells which areCD34+, or CD34+ and CD38−. It will be appreciated that it is alsopossible to use hematopoietic progenitor cells or stem cells capable ofgiving rise to cells which are CD34+, or more preferably CD34+ andCD38−. Preferably at least 10%, 20%, 50%, 75%, 90%, 95% or 99% of thecells, or essential all of the cells, in the cell compositionadministered to a mammal are hematopoietic progenitor or stem cells,and/or will express the CD34+ marker. The administered cells preferablycomprise cells capable of reconstituting the immune system in a lethallyirradiated host.

Most preferably, the cells are administrated to a subject by intravenousadministration. Optionally, a subject is pre-treated in order to enhanceengraftment of said progenitor or stem cells. Preferably, the cellsadministered to a subject are autologous cells.

As mentioned, typically the mammal to which the cells are administeredaccording to the invention is affected by or susceptible to beingaffected by a CNS disorder. The methods of the invention will preferablyresult in a reduction in the severity of central nervous system damageor symptoms of a central nervous system disorder in a mammal. In mostpreferred aspects, CNS disorders include Alzheimer's disease, or anyother CNS disorder characterized by diffuse neurodegeneration.

The methods and cells of the present invention can generally be used ina wide range of therapeutic applications. For example, cells may be usedin order to replace or enhance a factor normally present in the CNS of asubject. The replacement may be of a function carried out by a subject'snative microglia, as microglia are involved in many different biologicalfunctions, including examples as further discussed herein. However,cells of the invention are expected to be capable of expressinggenerally any suitable polypeptide such that replacement may also be ofsubstantially any function or activity normally present in the CNS orcarried out by a cell type present in the CNS. In other aspects, cellscan be used to inhibit a function carried out by a subject's nativemicroglia.

The invention further provides several advantageous therapeutic methodswhich can be carried out according to any of the methods ofadministering a nucleic acid or cell described herein. Disclosed in oneaspect is a method of treating a central nervous system disorder in amanual comprising: a) providing a hematopoietic progenitor or stem cell;and b) administering said composition to a mammal affected by orsusceptible to being affected by a CNS disorder, wherein saidhematopoietic progenitor or stem cell gives rise to cells characterizedby exhibiting decreased TNF-α secretion. In another aspect, theinvention provides a method of treating HIV, optionally HIV dementiacomplex in a mammal comprising: (a) providing a hematopoietic progenitoror stem cell capable of expressing a polypeptide selected from the groupconsisting of: a mutated form of a CCR5 receptor, a mutated form ofCXCR4, an CXCR4 ligand and a factor capable of inhibiting downstreamsignaling of CXCR4; and (b) administering said composition to a mammalaffected by or susceptible to being affected by HIV, optionally HIVdementia complex.

Also provided is a method of treating a neurodegenerative disease in amammal comprising (1) providing a hematopoietic progenitor or stem cell,preferably comprising a nucleic acid of interest; and (2) administeringsaid composition to a mammal affected by or susceptible to beingaffected by a neurodegenerative disease, wherein said hematopoieticprogenitor or stem cell migrates to the CNS and is capable of expressinga nucleic acid of interest in the CNS. Preferably the mammal to whichthe cells are administered is affected by or susceptible to beingaffected by CNS disease, such for example Alzheimer's disease. In oneembodiment, said hematopoietic progenitor or stem cell gives rise tocells capable of modulating inflammation, e.g. interrupting inflammatorysignaling cascades, in the CNS. In one embodiment, said hematopoieticprogenitor or stem cell gives rise to microglia characterized byinhibiting or inactivating the complement pathway. Preferably saidhematopoietic progenitor or stem cell comprises a nucleic acid ofinterest encoding a polypeptide acting as a C1 inhibitor. In anotherembodiment, said hematopoietic progenitor or stem cell comprises anucleic acid of interest encoding a polypeptide acting as a COX-2inhibitor. In other embodiments, said hematopoietic progenitor or stemcell gives rise to microglia capable of up regulating Aβ processing. Inyet other embodiments, said hematopoietic progenitor or stem cell givesrise to microglia capable of inhibiting the binding of Aβ peptides tomicroglia type-A macrophage scavenger receptors. In another embodiment,said hematopoietic progenitor or stem comprises a nucleic acid ofinterest encoding a neuronal trophic factor.

The invention also encomposses a method of treating a central nervoussystem disorder in a mammal comprising providing a hematopoieticprogenitor or stem cell; and administering said composition to a mammalaffected by or susceptible to being affected by a CNS disorder, whereinsaid hematopoietic progenitor or stem cell gives rise to cells capableof activating NF-κB signaling. In another aspect, said hematopoieticprogenitor or stem cell gives rise to cells capable of inhibiting NF-κBsignaling.

All the methods of administering a nucleic acid of interest or a cell tothe central nervous system of a mammal, particularly of a human,according to the present invention, methods which can be considered asmethod of treatment of an animal or a human body, could be converted asclaims of use of a nucleic acid of interest or a cell in the preparationof a composition or a medicament for the treatment of a mammal,particularly of a human, affected by or susceptible to being affected bya CNS disorder wherein the characteristics of the claims methods can beincluded without limitations.

So, in another aspect of the present invention, the invention alsocomprises the use of a nucleic acid of interest for the manufacture of acomposition for administration to a mammal, preferably a human, for thetreatment of a subject affected by or susceptible to being affected by aCNS disorder, wherein said composition is a composition enriched incells expressing the CD34 marker or cells capable of giving rise tocells expressing the CD34 marker, at least of portion of said cellscomprising a nucleic acid of interest, and wherein at least a portion ofsaid administered cells are capable of migrating to the CNS andexpressing the nucleic acid of interest in the CNS of this subject.

In a preferred embodiment, the present invention comprises the useaccording to the present invention, wherein said administered cells arecapable of giving rise to microglia in the CNS of said subject.

In still another aspect of the present invention, the present inventionrelates to the use of a nucleic acid sequence encoding a polypeptide ofinterest for the manufacture of a composition or a medicament foradministration to a mammal, preferably a human, for the treatment of asubject affected by or susceptible to being affected by a CNS disorderunder conditions that result in the expression of a polypeptide ofinterest at a level that provides a therapeutic effect in said subject,wherein said composition is a composition comprising hematopoieticprogenitor or hematopoietic stem cells which have been isolated fromcells comprising heematopoietic progenitor or stem cell obtained from asubject, and wherein a nucleic acid encoding a polypeptide of interesthas been introduced to said isolated hematopoietic progenitor or stemcell.

In still another aspect of the present invention, the present inventionrelates to the use of cells for the manufacture of a composition or amedicament for administration to a mammal, preferably a human, for thetreatment of a subject affected by or susceptible to being affected by aCNS, wherein said composition is a composition enriched in cellsexpressing the CD34 marker or cells capable of giving rise to cellsexpressing the CD 34 marker, and wherein at least a portion of saidadministered cells are capable of migrating to the CNS and giving riseto microglia

In a preferred embodiment, the present invention comprises the useaccording to the present invention, wherein said administration resultsin a reduction in the severity of central nervous system damage orsymptoms of a central nervous system disorder.

In still another aspect of the present invention, the present inventionrelates to the use of a nucleic acid sequence encoding a polypeptide ofinterest for the manufacture of a composition or a medicament foradministration to a mammal, preferably a human, for the treatment of asubject affected by or susceptible to being :affected by a CNS disorderunder conditions that result in the expression of a polypeptide ofinterest at a level that provides a therapeutic effect in said subject,wherein said composition is a composition enriched in cells expressingthe CD34 marker or cells capable of giving rise to cells expressing theCD34 marker, at least of portion of said cells being recombinant cellscomprising a nucleotide sequence encoding said polypeptide operablylinked to expression control elements.

In a preferred embodiment, the present invention comprises the use of anucleic acid sequence encoding a polypeptide of interest according tothe present invention, wherein at least a portion of said administeredcells migrate to the CNS, give rise to microglia and express the nucleicacid of interest in the CNS of said subject.

In a more preferred embodiment, the present invention comprises the useof a nucleic acid sequence or cells according to the present invention,wherein said administered cells expressing the CD34 marker, cellscapable of giving rise to cells expressing the CD34 marker,hematopoietic progenitor or hematopoietic stem cell differentiates intoa microglia cell.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence according to the present invention,wherein at least a portion of said administered cells express thenucleic acid of interest in the CNS of said subject.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein at least 20% of cells in said cell compositionexpress the CD34+ marker, preferably at least 50%, 90% or essentiallyall of cells in said cell composition ekpress the CD34+ marker.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein the administered cells are autologous to the subjectto be treated.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein administration is by intravenous administration.

In another more preferred embodiments the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein the subject to be treated is pretreated in order toenhance engraftment of said hematopoietic progenitor or hematopoieticstem cells.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said hematopoietic progenitor or hematopoietic stemcells or cells expressing the CD34+ marker are prior isolated.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said hematopoietic progenitor or hematopoietic stemcells are recombinant cells comprising a nucleic acid of interest.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said at least a portion of said hematopoieticprogenitor or hematopoietic cells are transduced with a vectorcomprising a nucleic acid of interest operably linked to a promotorcapable of effecting the expression of said nucleic acid of interest insaid cell.

Preferably, said at least a portion of said hematopoietic progenitorcells or hematopoietic stem cells are transduced with a viral vector,particularly with a lentiviral vector.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said hematopoietic progenitor or hematopoietic stemcells express the CD34+ marker or are capable of differentiating intocells expressing the CD34+ marker.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said cells are hematopoietic progenitor cells orhematopoietic stem cells.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said administered cells comprises cells capable, inan animal model, of reconstituting the immune system in a lethallyirradiated host.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said administered cells are human cells.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein said nucleic acid encodes a nonsecreted or a secretedprotein.

In another more preferred embodiment, the present invention comprisesthe use of a nucleic acid sequence or cells according to the presentinvention, wherein the CNS disorder which affects or which issusceptible to affect the subject is characterized by diffuseneurodegeneration, such for example Alzheimer's disease.

DESCRIPTION OF THE FIGURES

FIGS. 1A to 1B: Phenotype and expression of ALD protein in bone marrowfrom NOD-SCID mouse transplanted with human ALD deficient CD34+genetically engineered to express the ALD protein.

Cells were phenotyped by flow cytometry using monoclonal antibodiesagainst human CD45 (FIG. 1A), CD19, CD15 (FIG. 1B), CD14 and CD11 (FIG.1C) surface antigen markers.

FIG. 2: Bone marrow from NOD-SCID mouse transplanted with human ALDdeficient CD34+ cells genetically engineered to express the ALD proteincontains CD34+CD38− cells, indicating that early human hematopoieticprogenitor cells were maintained in vivo.

FIGS. 3A to 3C: In situ hybridization of brain from NOD-SCID mousetransplanted with human ALD deficient CD34+ genetically engineered toexpress the ALD protein.

Cells containing human alu DNA sequences are present in the white matterof corpus callosum (FIG. 3A, arrow) and in the cerebellum (FIG. 3B,arrows). Cells strained with microglia marker (RCA, in green,fluorescein) express ALD protein (Cy3 in red) (FIG. 3c).

DETAILED DESCRIPTION

Microglia

In the brain parenchyma, macrophages are called microglia. They werefirst recognized by Rio Hortega in 1932. Brain parenchymamacrophages/microglia are quite distinct from neurons, other glial cells(astrocytes, oligodendrocytes) and also distinct from macrophagesassociated with other part of the CNS (leptomeninges, choroid plexus,perivascular macrophages).

Microglia comprise a significant proportion of the nonneuronal cellpopulation in the CNS: 5% in the white matter, up to 2% in the greymatter; up to 10-20% of all glial cells. Microglia are present in bothgrey and white matter. Some variation exists in the number of microgliacells among different brain regions but this does not reach one order ofmagnitude (Dobrenis K. Methods in Enzymology, 6:320-344, 1998; Perry V Hand Gordon S. Trends Neurosci., 11:273-277, 1988; Perry V H. and GordonS. Int. Rev. Cytol., 125:203-244, 1991; Perry V H. Macrophages in thecentral nervous system pp 87-101, R G Landes, Austin, 1994;Gonzales-Scarano F and Baltuch G. Annu. Rev. Neurosci., 22:29-240, 1999;Mittelbroon M. et al., Acta Neuropathol., 101:249-255, 2001).Perineuronal microglia are cells with somata that abut that of aneuronal perikaryon, often intimately wraps or covers a portion of theneuronal cell body.

Several markers recognize microglia including antibodies against surfaceantigens (HLA-DR; CD11a, CD11b and CD11c which are members of the β2integrin family; leukocyte common antigen, Fcγ receptor, F4/80, MAC-1)and lectins (Griffonia simplicifolia I-B4). There is however no markerthat specifically recognizes brain microglia and not macrophages locatedin other tissues. Adult microglia is often referred as “quiescent” or“resting” microglia, distinguishing it from “activated” microglia thatarise in many pathological states. Resting microglia is ramified anddownregulates the expression of most antigenic markers (ED1, CR3complement receptor, MHC antigens) and functional indicators (cytokines)associated with macrophages in other tissues (Dobrenis, 1988, supra;Perry V H and Gordon S, 1988, supra; Perry V H and Gordon S, 1991,supra; Perry V H, 1994, supra; Gonzales-Scarano and Baltuch G, 1999,supra). In contrast to other organs where differentiated macrophagesoutnumber colonizing precursors, the majority of microglia (restingmicroglia) remains in an undifferentiated state towards immunologicresponse (Santambrogio L et al., Proc. Natl. Acad., Sci. USA.,98:6295-6300, 2001). When resting microglia becomes activated, theramified appearance begins to withdraw, the cell body enlarges and cellreenters the cell cycle to undergo mitotic division.

Origin of Microglia

In contrast to other glial cells (astrocytes, oligodendrocytes) that arederived from neuroectoderm, observations have supported that microgliahave a myelomonocytic origin. In particular, it has been observedthat: 1) cells from bone marrow enter the CNS and adopt the morphologyof microglia; 2) that monocytes invade the developing CNS and cantransform to microglia (Hume D A et al., J. Cell Biol. 97:253-257,1983;.Perry V H., Pontif. Acad. Sci. Scr. Varia, 59:281-295, 1985); 3)that microglia express antigens known to be partly or wholly restrictedto cells of the monocytic lineage.

Evidence that bone-marrow derived cells enter the CNS was obtained frombiochemical and histochemical analysis after bone marrow transplantation (BMT) in mice and rats (Ting J P et al., Immunogenetics,17:295-301,1983; Hickey W F and Kimura H. Science 239:290-292, 1988;Hickey W F et al., J. Neuropathol. Exp. Neurol., 51:246-256, 1992;Hoogerbrugge P M et al., Science 239:1035-1038, 1988; DeGroot, 1992;Lassmann H. and Mickey W F., Clin. Neuropathol., 12:284-285, 1993a;Lassmann H. et al., Glia 7:19-24, 1993b; Krall W J et al., Blood9:2737-2748, 1994; Krivit W et al,. Cell Transplantation., 4:385-391,1995; Eglitis M A and Mezey E. Proc. Natl. Acad. Sci. USA.,94:4080-4085, 1997). In these transplantation studies, donor bone marrowcells carried genes foreign for the donor, including ones for MHC,lysosomal enzyme, E. Coli galactosidase, SrY and λ phage.

Among these above referenced documents, the document Krall W J et al.,(Blood 9:2737-2748, 1994) can be particularly cited. This publicationdiscloses the study of macrophage and microglia replacement after murineautologous bone marrow transplantation with retrovirus-marked bonemarrow. The authors indicate that, in the brain, 20% of the totalmicroglia had been replaced with donor cells expressing the humangiucocerebrosidase (GC) by 3 to 4 months after transplant.

Bone marrow transplantation (BMT) in rodents leads to a relatively rapidturnover of non parenchymal macrophages (20-40% turnover of perivascularmacrophages 3 months after BMT). Turnover of resting and ramifiedmicroglia is slower (5 to 20% among different studies, 3 months afterBMT). The turnover of macrophages is not restricted to perivascularmacrophages as donor-derived ramified microglia has clearly beenidentified after bone marrow transplantation.

Most studies showing that bone marrow derived cells can differentiateinto microglia were performed in rodents. For evident reasons, there arefew data showing that the same process can occur when using human bonemarrow cells. However it has been clearly demonstrated that male donorbone marrow derived cells can be recovered in the brain of female humanbrain after bone marrow transplantation (Unger E R et al., J.Neuropathol. Exp. Neurol., 52:460-470, 1993).

Myelomonocytic cells (and lymphocytes) are known to enter into the CNSvia several routes: the lepto-meninges, choroid plexus and perivascularareas surrounding small vessels. The entry of these cells into CNS canbe enhanced when the blood-brain-barrier is disrupted or modified, as itoccurs when “inflammatory” changes take place into CNS (Dobrenis, 1988,supra; Perry V H and Gordon S; 1988, supra; Perry V H and Gordon S,1991, supra; Perry V H, 1994, supra; Gonzales-Scarano and Baltuch, 1999,supra).

Nevertheless, despite evidence suggesting that 1) monocytes can enterthe brain and differentiate in perivascular macrophages and; 2) cellsderived from the donor and having the morphological andhisto-immunochemical characteristics of microglia can be recovered inthe recipient mice after bone marrow transplantation, until the presentinvention, one did not know at which stage of differentiation (fromearly primitive HSC to already differentiated monocytic stage),hematopoetic cells can enter into the brain after bone marrowtransplantation and differentiate into microglia. Thus, previousexperiments demonstrating in mice that brain microglia cells are derivedfrom bone marrow cells have been performed using transplantation oftotal bone marrow cells. Using total bone marrow for treatment, however,presents significant disadvantages.

Functions of Microglia

Microglia interact with neurons, astrocytes and oligodendrocytes as wellas extracellular elements in the CNS. Microglia have many functions(Perry V H and Gordon S., 1988, supra; Perry V H and Gordon S., 1991,supra; Perry V H, 1994, supra; Gonzales-Scarano and Baltuch, 1999,supra) several of which are further described as follows.

Among the known functions of microglia are important roles inphagocytosis, extracellular matrix catabolism and the production ofgrowth factors during development as well as in the adult CNS. Thusmicroglia participate in the modeling of the CNS during the developmentand also act in a neuroprotective way against several types of injuries.

Microglia also have an important role in homeostasis. Microglia produceneurotransmitters and neuropeptides that interact with neurons and otherglial cells.

Furthermore, microglia are involved in lipid turn-over, includingganglioside and phospholipid catabolism, a polipoprotein binding andsecretion.

Microglia are also involved in inflammation, where activated microgliarelease cytokines (TNF-α, interferons, IL-1, IL-6), complement proteins,arachidonic acid (that potentiates NMDA receptor currents in neurons),:chemokines, cysteine, quinolinate, the amine Ntox which also potentiatesNMDA receptor activation, neutral proteases, oxidative radicals, andnitric oxide that may contribute to death of neurons in severaldiseases. However, depending on the magnitude, timing and type ofstimulus, activated microglia can also contribute to host defense andrepair (Minghetti L. and Levi G. Prog. Neurobiol., 54:99-125, 1998;Gonzales-Scarano and Baltuch, 1999, supra; Akiyama et al., 2000). Thus,IL-6 plays a key role in regulating neuronal survival and function. IL-6may cooperate with the high affinity neurotrophin receptor Trk. IL-6 canalso act as an indirect immunosuppressant because it stimulates thepituitary-adrenal axis and elicits release of glucocorticoids. IL-6 alsoinhibits interferon-γ IL-1β and LPS (liposaccharide) induced synthesisof TNF-α (Akiyama et al., 2000). TNF-α may be. cytotoxic in braintrauma, multiple sclerosis and ischemic injury, but TNF-α can be trophicto rat hippocampal neurons, protects against glutamate, free radical andAβ toxicity in cultured neurons. TNF-α, and is a potent stimulator ofNF-κB, a transcription factor that increases the expression of survivalfactors such as calbindin, manganese-superoxide dismutase and theanti-apoptotic Bcl-2 protein (Akiyama et al., 2000).

Microglia are also involved in the immune response. Microglia are theprincipal immune cells in the CNS and play a role in antigen processing(APC-like cells). Microglia respond to traumatic injury or the presenceof pathogens by migrating to the site of injury where they becomeactivated and may proliferate. Like other macrophages, microglia releasecytokines that rectute other cells (T and B cells) to the site ofinjury.

By providing cells that can give rise to microglia upon administrationto a subject, any of these functions or properties of microglia can beprovided, enhanced or modified to a subject in need thereof bydelivering microglia to the CNS according to the invention. The functioncan be provided by administering unmodified cells (e.g. allogeneic)according to the methods of the invention thereby taking advantage ofmicroglia's normal therapeutic capacities, or the function may beprovided, enhanced or modified by administering cells which have beenmodified by the introduction of a therapeutic nucleic acid. As will beappreciated and further described herein, the introduction of a nucleicacid may be also useful to deliver a function not normally performed bymicroglia.

Dual Role of Microglia in the Pathogenesis of Neurodegenerative Diseases

According to the preferred methods of the present invention, microgliacan be exploited in a therepeutic treatment in order to benefit fromeither or both of their dual roles in neurodegenerative disease.Preferred examples, further discussed below as well as in the sectiontitled “Treatment”, include methods of treating neurodeneration such asin the exemplary cases Alzheimer's disease, Parkinson's disease,multiple sclerosis, and HIV dementia complex as well as inneuroprotection. Microglia may have deleterous or beneficial effects onthe progression of several neurodegenerative diseases. Two examples aregiven as paradigms: CNS infection due to HIV and Alzheimer's disease.

HIV Infection

Individuals with HIV infection are predisposed to develop anopportunistic infections (toxoplasmosis) and neoplasmas (primarycerebral lymphoma) in the CNS. In addition, 20% of HIV-infectedindividuals develop a neurological syndrome, referred to as AIDSdementia complex or HIV dementia (HIVD), consisting of motordysfunction, cognitive deterioration, and in later stages coma. Thisneurological syndrome is caused by HIV infection itself. In HIVD, thereis scant evidence of infection of neurons. HIV enters the CNS viacirculating lymphocytes or monocytes, which in turn transmit the virusto perivascular macrophages-microglia. Infected microglia survive forlong periods of time and produce enough virus to maintain a cycle of newinfections. Microglia express the β-chemokine receptor CCR5, which isthe primary co-receptor for HIV (M-tropic isolates) with CD4. CCR5 isalso the natural receptors for chemokines MIP-1α, MIP-1β and RANTES.Microglia express also CXCR4 (whose natural ligand is chemokine SDF-1)which can be used by HIV (SI isolates) to enter into these cells(Gonzales-Scarano and Baltuch, 1999, supra). In addition to their rolein maintenance of infection of HIV within the brain, microglia arelikely to have a direct role in neurotoxicity observed in HIV dementia.Among the candidate proteins secreted by HIV-infected microglia, thecoat protein gp120 plays a role in activating indirectly NMDA receptorson neurons that leads to calcium influx and neuronal death (Bezzi P. etal., Nat. Neurosci., 4, 702-710, 2001). One role of microglia, and theTNF-α released by them, is to potentiate prostaglandin-dependentglutamate release from astrocytes that will activate NMDA receptors onneurons. Binding of gp120 on CXCR4 receptors at the surface of microgliaevokes a large release of TNF-α which acts on the astrocyte signallingpathway to increase the production of prostaglandins (PgE2) and henceglutamate in the extracellular space.

Alzheimer Disease (AD)

Alzheimer disease (AD), the major cause (70%) of dementia in adult is aprogressive neurodegenerative disorder that occurs in 5% of thepopulation over 65 years of age. It is clinically characterized by aglobal decline in memory and other cognitive functions that leavesend-stage patients bedridden, incontinent and dependent on custodialcare. Death occurs on average nine years after the diagnosis. The majorrisk for AD is increasing age and in the USA alone, there are currentlyover four millions patients with AD.

The major neuropathological changes in the brain of AD patients areneuronal death, particularly in regions related to memory and cognitionand the presence of abnormal intra- and extra-cellular proteinaceousfilaments. Intracellularly, bundles of paired helical filaments (PHF),composed largely of phosphorylated tau protein and referred to asneurofibrillary tangles, accumulate in large number in dying neurons.Extracellularly, insoluble aggregates of proteinaceous debris, termedamyloid, appear in the form of senile or neuritic plaques andcerebrovascular amyloid deposits. The amyloid deposits consist ofaggregates of amyloid β-peptide (Aβ) isoforms. These are 39-42 residuepeptides that are proteolytically derived from the large amyloidprecursor (APP) by two proteases, β-secretase and α-secretase, andsecreted by all cells. Cells secrete more Aβ40 than Aβ42 isoform that isless soluble and forms the major component of amyloid plaques. The factthat mutations in the APP gene are associated with familial AD is astrong indication of the importance of amyloid in the pathogenesis ofthe disease. The observation that activated microglia cluster aroundsenile plaques suggests that microglia play an important role in thedisease pathogenesis (Gonzales-Scarano and Baltuch, 1999, supra;Weninger S C and Yankner B A., Nat. Medecine, 7:527-528, 2001).Microglia in AD may have both deleterious and beneficial effects.Fibrillar Aβ peptides stimulate microglia leading to COX-2 activation,release of cytokines (TNFα, IL-1β, IL-6) and complement proteins thatcontribute to neurodegeration (Akkiyama et al., Neurobiology of aging.21:383-421, 2000). High concentrations of Aβ40 or Aβ42 peptides do notdamage neurons unless microglia are present. The HHQK domain within Aβpeptide provides a recognition site for microglial binding (Giulian D.,Am. J. Hum. Genet., 65:13-18, 1999). However, microglia may have abeneficial effect in removal of the neurotoxic Aβ peptides. Microgliainternalize Aβ fibrils by a type-A macrophage scavenger receptor(Paresce D M et al., Neuron 17:553-565, 1996), which is stronglyexpressed on activated microglia in the vicinity of senile plaques. Thedegradation of Aβ protein by microglia occurs via a secreted nonmatrixmetalloprotease. The rate of Aβ degradation by microglia is howeverlimited and the cells may be overwhelmed by the amount of Aβ present. Inaddition, it is not impossible that Aβ itself stimulates microglia toproduce more Aβ by an autocrine loop.

Microglial Transplant as a Therapeutic Modality

The lineage of monocytes, microglia, and brain macrophages offer asimple and effective strategy for delivery of agents to the CNS in aglobal manner. As discussed herein, monocytes normally enter the CNS.This occurs during development but also in adulthood. Bone marrowtransplantation experiments in rodents have demonstrated that turnoverof parenchymal and resting microglia occurs, albeit at a slower ratethan that for perivascular macrophages. Given the high degree ofvascularization of the CNS, entry of microglia precursors can occur in awidespread manner and migrate into grey and white matter. The entry ofthese cells into CNS can be enhanced when the blood-brain-barrier isdisrupted or modified, as it occurs when “inflammatory” changes takeplace into CNS.

Allogenic bone marrow transplantation has been used in humans to treatseveral genetic disorders that affect the CNS (Krivit W. et al., CellTransplantation, 4:385-391, 1995; Krivit W. et al., Cur. Opin.Hematology, 6:377-382, 1999). The first group includes several lysosomalstorage disorders with widespread CNS involvement. In these disorders,deficiency of a lysosomal enzyme affects primarily neurons (as inHurler's disease) or oligodendrocytes (as in metachromaticleukodystrophy or Krabbe disease). The rationale for proposing BMT isthese disorders is that monocyte-derived cells from the donor, can enterthe brain, differentiate into microglia and/or perivascular macrophagesand secrete a normal lysosomal enzyme that can be recaptured by neuronsor oligodendrocytes lacking this lysosomal enzyme. BMT was shown to beefficacious in an other genetic CNS disorder, X-linkedadrenoleukodystrophy (ALD) (Aubourg P. et al., N. Engl. J. Med.,323:1860-1866, 1990; Shapiro E. et al., Lancet 356:713-718, 2000). Thisdisorder is characterized by progressive and widespread demyelinationwithin the CNS, but in contrast to lysosomal storage disorders, the ALDgene encodes a non-secreted protein localized in the membrane of anintracellular organella (the peroxisome), which is a member ofATP-binding cassette transporter superfamily. Long-term efficacy of BMThas been confirmed in several CNS lysosmal storage diseases and ALD.Thus, replacement of endogenous microglia by normal donor-derivedmicroglia can cure or halt CNS diseases characterized by widespreadneuronal or glial pathology. BMT may allow the correction of CNS diseasevia two mechanisms: 1) the secretion of a “therapeutic” protein which islacking or defective in neurons or other glial cells; 2) the replacementof endogenous defective nucroglia by microglia with normal function.

Efficacy of allogenic BMT is however markedly limited by the lack ofHLA-identical donor and is associated with a significant mortality riskwhich is mainly due to rejection of the graft and/or severegraft-versus-host disease (GVH). Autotransplantation of hematopoieticstem cells (HSC) would circumvent these problems. In addition, since HSCcan be genetically modified ex vivo prior to reinfusion. (Somia N. andVerma I M., Nature Rev., 1:91-99, 2000; Kay M A et al., Nat. Medecine.7:33-40, 2001), any relevant “therapeutic” protein can be produced byHSC-derived microglia, in particular proteins that enhance CNS defenseand repair. In addition, it is envisioned that HSC can be geneticallymanipulated in order to engineer microglia in which activation that maybe deleterious in several CNS diseases is avoided.

In contrast to neural stem cells that are well characterized, primaryHSC have not yet been fully characterized (in human as well as inmouse). However, various subpopulations of hematopoeitic cells from bonemarrow containing HSC have been isolated, based on the presence/absenceof antigen marker(s) on their surface. Several of the antigens andmethods for obtaining enriched cell compositions or isolated cells arefurther described herein. This includes the sialomucin CD34 marker whichallows the recovery of primitive HSC from bone marrow or from peripheralblood after stimulation with G-CSF (Krause D S et al., Blood 87:1-13,1996). Allogenic transplantation or auto-transplantation of CD34+ cellsare routinely performed in human patients and all relevant clinical andexperimental protocols are designed, for CD34+ cells enriched by avariety of selection methods (Krause et al., 1996, supra). In rodents,long-term repopulation assays indicate that some stein cells that do notexpress detectable levels of CD34 antigen are also able to reconstitutebone marrow after transplantation in lethally irradiated recipientanimals. This includes cells selected by high efflux of Hoechst 33342dye (Goodell M A. et al., Nat. Medecine 3:1337-1345, 1997), by ALDHexpression (Jones R J. et al., Blood 88:487-491, 1996), and CD34−/SRCcells (Bhatia M. et al., Nat. Medecine, 4:1038-1044). The evidence thatCD34 negative cells also represent a population of HSC has however notbeen demonstrated in human.

As murine HSC and human CD34+ cells can be genetically modified ex vivo(for example after transduction with retrovirus or HIV-1 derivedlentivirus vectors) (Case S C et al., Proc. Natl. Acad. Sci. USA,96:2988-2993, 1999; Somia and Verma, 2000, supra; Kay et al., 2001,supra; Douglas J L et al., Hum. Gen. Ther., 12:401-413, 2001), thepresent invention provides the transplantation of human CD34+ cells thathave been genetically modified ex vivo with the aim to express one ormore specific transgenes in the microglia after transplantation.Additionally support for the feasibility is provided by Krall et al.(1994), supra, demonstrating that up to 20% of the total microglia ofmouse brain can be replaced with donor cells expressing the humanglucocerebrosidase enzyme (GC, a lysosomal enzyme which is deficient inthe human disorder called Gaucher disease) after transplantation ofsyngenic bone marrow cells that were previously transduced ex vivo witha retroviral vector expressing the human GC. Additionally, data fromEglitis (1997), supra, demonstrated that microglia cells can express atransgene (the neomycine gene) after transplantation of bone marrowcells that were transduced ex vivo prior to transplantation.

Thus, according to the present invention, similar ex vivo geneticmanipulation can be performed using compositions of cells that areenriched in or contain isolated populations of hematopoietic stem orprogenitor cells for the delivery of a cell or polynucleotide to thebrain. As described above and in the Examples, the present inventorshave shown that human CD34+ cells and derived myelomonocytic cells canenter into the brain, differentiate into microglia and express atherapeutic protein. CD34+ cells from ALD patients were transduced witha HIV-1 derived vector carrying the ALD cDNA. ALD CD34+ cells wereobtained from plasmapheresis after G-CSF was administered to patients.Up to about 50% of ALD deficient CD34+ cells were transduced by theHIV-derived lentiviral vector and expressed the ALD protein (all ALDCD34+ cells were ALD protein negative before the transduction owing toALD gene mutation). The lentiviral-vector encoded ALD protein was shownbiochemically to be functional in peroxisomes of transducedhematopoietic ALD cells by assessing the accumulation of VLCFAs, adeficiency caused by lack of functional ALD protein. These geneticallymodified human CD34+ cells as well as normal cord blood human CD34+cells were transplanted (as a xenograft) into SCID-NOD mice. These micehave a severe combined immunodeficiency and transplantation of wholehuman bone marrow cells or human CD34+ cells was previously shown toreconstitute partially a hematopoietic cell system in these mice. Micewere engrafted with the transduced ALD deficient CD34+ cells inproportions ranging from 25% to 75% (% age of donor derived cellsrecovered in the bone marrow), and CD34/CD38− cells were found,indicating that early human hematopoietic progenitor cells weremaintained in vivo. CD34+ cells from a tranplant recipient were alsoshown to contain CD68 positive cells expressing ADLP, indicating thatlong-term NOD/SCID repopulating cells derived from transduced ALDdeficient CD 34+ cells were able to differentiate intomonocytes/macrophages and express recombinent ALDP in bone marrow.Importantly, ALD positive cells were also recovered in the brain of thetransplanted SCID-NOD mice. These cells expressed the donor-derivedhuman Y chromosome, had the morphology of perivascular macrophages orramified microglia and expressed RCA, a well recognized marker formicroglia. ALD positive human microglia cells derived from normal cordblood human CD34+ cells or transduced ALD CD34+ cells were present inthe grey and white matter of the SCID-NOD mice. ALDP was expressed inthis way by human brain microglia for up to 4 months.

Definitions

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc. The term includescloning and expression vehicles, as well as viral vectors.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

A “coding sequence” or a sequence which “encodes” a particular protein,is a nucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom prokaryotic or eukaryotic mRNA, genonic DNA sequences fromprokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

The terms DNA “control sequences” or “expression control element” referto promoter sequences, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, origins ofreplication, internal ribosome entry sites (“IRES”), enhancers, and thelike, which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesecontrol sequences need always be present so long as the selected codingsequence is capable of being replicated, transcribed and translated inan appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleic acid region-comprising a DNA regulatory sequence, whereinthe regulatory sequence is derived from a nucleic acid sequence which iscapable of binding RNA polymerase and initiating transcription of adownstream (Y-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition. Forthe purpose of describing the relative position of nucleotide sequencesin a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream”, “downstream” relative to another sequence,it is to be understood that it is the position of the sequences in the“sense” or “coding” strand of a DNA molecule that is being referred toas is conventional in the art.

By “isolated” when referring to a hematopoietic stem cell or progenitorcell, is meant that the indicated cell or cell type having a specifiedfeature is present in the substantial absence of other cells not havingsaid feature. Thus, an “isolated hematopoietic cell expressing a CD34molecule” refers to a cell which is substantially free of otherhematopoietic cells or different cell types that do not express the CD34molecule; however, a composition of isolated cells may include someadditional cells, so long as do not deleteriously affect the basiccharacteristics of the composition. Said isolated cell or cellcomposition may also include some cells of a different type as long assaid cells express a specified feature, e.g. express a CD34 molecule.

The term “purified” does not require absolute purity; rather, it isintended as a relative definition. Purification of cells having aspecified characteristic to at least one order of magnitude, preferablytwo or three orders, and more preferably 10, 100, 200 or 1000 orders ofmagnitude over that of a natural source of the cells is expresslycontemplated. The term “purified” is further used herein to describe acell or cell composition of the invention which has been separated fromother cells not having a specified characteristic (e.g. hematopoietictype, a cell surface marker, progenitor cell, etc.). A cell compositioncan be said to be substantially pure when at least about 50%, preferably60 to 75%, more preferably at least about 80, 90, 95 or 99% of the cellsin a sample exhibits a specified characteristic.

The term “hematopoietic progenitor cell” , as used herein, refers to anundifferentiated cell derived from a hematopoietic stem cell, and is notitself a stem cell. Some progenitor cells can produce progeny that arecapable of differentiating into more than one cell type. Adistinguishing feature of a progenitor cell is that, unlike a stem cell,it has limited proliferative ability and thus does not exhibitself-maintenance. It is committed to a particular path ofdifferentiation and will, under appropriate conditions, eventuallydifferentiate into one of various cell types.

A “stem cell”, also referred to as a “pluripotent stem cell”, may bedefined by its ability to give rise to progeny in all defined lineages.Stem cells are the multipotent self-renewing cells that sit at the topof the lineage heirarchy and proliferate to make differentiate cellstypes of a given tissue in vivo. Hematopoietic stem cells possessed theability to fully reconstitute the immune system of a lethally irradiatedhost from which the cells are obtained. The hematopoietic stem cellsgive rise to all blood and immune cells. However, recent data suggestthat stem cells from a given organ can also give progeny to cells thatdifferentiate into cells from another organ, provided that the stemcells are in the appropriate microenvironment. Thus, bone marrow cellsthat contain hematopoietic stem cells can contribute to, astrocytes andneurons in the brain, skeletal muscle cells in tibialis anterior(Gussoni, E. et al., Nature, 1999, 401(6751):390-4; and Ferrari, G. etal., Science, 1998, 279(5356):1528-30), hepatic oval cells orhepatocytes in liver (Petersen, Science, 1999, 284(5417):1168-70; andLagasse, E. et al., Nat. Med., 2000, 6(11):1229-34). Lin7⁻ c-kit^(POS)bone marrow cells can contribute to regeneration of myocytes ininfarcted myocardium (Orlic, D. et al., Nature, 2001, 410(6829):701-5).Bone marrow derived circulating cells have the capacity to be a sourceof intimal smooth muscle like cells in murine allograft aortictransplant (Shimizu, K. et al., Nat. Med., 2001, 7(6):738-41).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of, microbiology, molecular biology,recombinant DNA techniques and virology within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al., Molecular Cloning: A Laboratory Manual (CurrentEdition); Current Protocols in Molecular Biology (F. M. Ausubel, et al.,eds., current edition); DNA Cloning: A Practical Approach, vol. I & 11(D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., CurrentEdition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds.,Current Edition); Transcription and Translation (B. Hames & S. Higgins,eds., Current Edition); CR C Handbook of Parvoviruses, vol. I & 11 (P.Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & 11 (B. N.Fields and D. M. Knipe, eds.).

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural references unless the contextclearly dictates otherwise.

Obtaining Cell Populations

In contrast to neural stem cells that are well characterized, primaryhematopoietic stem cells (HSC) have not yet been fully characterized (inhuman and mouse). As best, various subpopulations of hematopoeiticprogenitor cells from bone marrow that contain HSC have been isolated,based on the presence/absence of antigen markers at their surface.

As used herein in the context of compositions enriched in hematopoieticprogenitor and stem cells, “enriched” indicates a proportion of adesirable element (e.g. hematopoietic progenitor and stem cells) whichis higher than that found in the natural source of the cells. Ingeneral, a natural source of cells will be processed so as to add orincrease the proportion of the hematopoietic progenitor and stem cells.A composition of cells may be enriched over a natural source of thecells by at least one order of magnitude, preferably two or threeorders, and more preferably 10, 100, 200 or 1000 orders of magnitude.Compositions enriched in hematopoietic progenitor or stem cells, orisolated hematopoietic progenitor or stem cells can be obtained foradministration to a particular subject can be autologous cells orallogeneic cells. Hematopoietic progenitor or stem cells can also bederived from fetal or embryonic human tissue that is processed and/orcultured in vitro so as to increase the numbers or purity of primitavehematopoietic elements. In humans, CD34⁺ cells can be recovered frombone marrow or from blood after cytokine mobilization effected byinjecting the donor with hematopoietic growth factors such asGranulocyte colony stimulating factor (G-CSF), granulocyte-monocytecolony stimulating factor (GM-CSF), stem cell factor (SCF)subcutaneously or intravenously in amounts sufficient to cause movementof hematopoietic stem cells from the bone marrow space into theperipheral circulation. Initially, bone marrow cells may be obtainedfrom any suitable source of bone marrow, e.g. tibiae, femora, spine,fetal liver, and other bone cavities. For isolation of bone marrow, anappropriate solution may be used to flush the bone, which solution willbe a balanced salt solution, conveniently supplemented with fetal calfserum or other naturally occurring factors, in conjunction with anacceptable buffer at low concentration, generally from about 5 to 25 mM.Convenient buffers include Hepes, phosphate buffers, lactate buffers,etc.

Cells can be selected using commercially available antibodies which bindto hematopoietic progenitor or stem cell surface antigens, e.g. CD34,using methods known to those of skill in the art. For example, theantibodies may be conjugated to magnetic beads and immunogenicprocedures utilized to recover the desired cell type. The CD34 antigen,which is found on progenitor cells within the hematopoietic system ofnon-leukemic individuals, is expressed on a population of cellsrecognized by the nonoclonal antibody My-10 (i.e., express the CD34antigen) and can be used to isolate stem cell for bone marrowtransplantation. See Civin, U.S. Pat. No. 4,714,680, the disclosure ofwhich is incorporated herein by reference. My-10 has been deposited withthe American Type Culture Collection (Rockville, Md.) as HB-8483 and iscommercially available from Becton Dickinson Immunocytometry Systems(“BDIS”) as anti-HPCA 1. However, using an anti-CD34 monoclonal antibodyalone is not sufficient to distinguish between true pluripotent stemcells and other more differentiated cells, since B cells (CD19⁺) andmyeloid cells (CD33⁺) make up 80-90% of the CD34⁺ population. Thus toimprove progenitor or stem cell selection, a combination of monoclonalantibodies can advantageously be used to select human progenitor andstem cells. It is also possible to isolate CD34⁺ cells from monkeys.

Another antigen which may be used in selection is Class II HLA(particularly a conserved DR epitope recognized by a monoclonal antibodydesignated J1-43). HLA-DR is found on progenitor cells (although not onstem cells), and thus provides for some enrichment of progenitoractivity by selecting for the marker, or for stem cells by negativeselection. While these markers are also found in numerous lineagecommitted hematopoietic cells, they nevertheless allow at least a firstimproved enriched population of cells to be obtained.

The Thy-1 antigen can also be used for selection. Thy-1 is expressed onboth progenitor cells and stem cells, and a particular subset of bonemarrow cells meeting the criteria for stem cells has been found toexpress low levels of Thy (Thy^(lo)) (Baum et al., PNAS 89:2804-2808,1992; Craig et al., 1993, J. Exp. Med 177: 1331-1342. A furtherselection antigen is c-kit, which is expressed on both hematopoieticstem and progenitor cells, although expression is gradually decreasedupon maturation (Ogawa et al, 1991, J. Exp. Med 174: 63-71). Thesedisclosures are incorporated herein by reference.

A sub-population of CD34⁺/CD38⁻ cells that contains more primitive HSChas been identified (Terstappen, 1991, Blood 77:1218-1227; Terstappen etal., 1994, Blood Cells, 20:45-63; Sutherland, 1989, the disclosures ofwhich are incorporated herein by reference). Preferably, humanhematopoietic stem cells are selected as being CD34+, CD38− incombination with lack of expression of the HLA-DR antigen (Verfaillie etal., 1990, J. Exp. Med., 172:509-520, the disclosure of which isincorporated herein by reference). While CD38 is expressed on 95-99% ofbone marrow derived CD34+ cells, the CD38− fraction forms colonies withlong term repopulating ability allowing a further purification ifdesired.

Additionally, a subpopulation of CD34⁻ cells that are positive for thedye Hoechst 33342 (Goodell M A et al., 1997, supra, the disclosure ofwhich is incorporated herein by reference, was also shown to containprimitive HSC since their transplantation is able to reconstitute bonemarrow in a host.

In mice, different markers are used: Lin−, Sca-1+, c-kit− and WGA forstem cells and Sca-1−, c-kit+ and WGA in progenitor cells. WGA, wheatgerm agglutinin, is also expressed by both progenitor and stem cells,and again can be used to discriminate between progenitor and stem cells.Hematopoietic stem cells are WGAdim and hematopoietic progenitor cellsare WGAbright (Ploemacher et al., 1993, Leukemia 7:120-130), Sca-1, stemcell antigen-1, is expressed on murine hematopoietic stem cells (Uchidaand Weissman, 1992, J. Exp. Med 175:175-184) and to a lesser extent onprogenitor cells (Spangrude et al., 1988, Science 241:58-62; andSpangrude et al., 1994, Ann. Rev. Med. 45:93-104). However, Sca- cellshave been shown to have a short term repopulating ability when injectedinto sublethally irradiated mice suggesting that Sca- may be used toselect committed hematopoietic progenitor cells. C-kit, mentioned above,can also be used for the selection of murine cells. Thy-1 also serves asa marker in mice and rats as well as in human. It has also been shownthat rhodamine-123 can be used to distinguish stem cells from progenitorcells (stem cells appear dull when stained while progenitors are bright)(Baum et al., 1992; Fleming et al., 1993, J. Cell. Bio 122:897-902;Chaudhary and Robinson, 1991, Cell 66:85-94). The above disclosures areincorporated herein by reference.

In one example, a combination of anti-CD34 and anti-CD38 monoclonal.antibodies can be used to select those human progenitor stem cells thatare CD34⁺ and CD38⁻. One method for the preparation of such a populationof progenitor stem cells is to stain the cells with immunofluorescentlylabeled monoclonal antibodies. The cells then may be sorted byconventional flow cytometry with selection for those cells that areCD34⁺ and those cells that are CD38⁻. Upon sorting, a substantially purepopulation of stem cells results. (Becton Dickinson Company, publishedEuropean Patent Application No. 455,482, the disclosure of which isincorporated herein by reference).

Additionally, negative selection of differentiated and “dedicated” cellsfrom human bone marrow can be utilized, to select against substantiallyany desired cell marker. In known examples, this technique has yielded apopulation of human hematopoietic progenitor or stem cells with fewerthan 5% lineage committed cells. See Tsukamoto et al., U.S. Pat. No.5,061,620, the disclosure of which is incorporated herein by reference.For example, progenitor or stem cells, most preferably CD34+ cells, canbe characterized as being any of CD3⁻, CD7⁻, CD8⁻, CD10⁻, CD14⁻, CD15⁻,CD19⁻, CD20⁻, CD33⁻, Class II HLA⁺ and Thy-1⁺.

Furthermore, a two-step purification of low density human bone marrowcells by negative immunomagnetic selection and positive dual-colorfluorescence activated cell sorting (FACS) can be used. In one example acell fraction was obtained that enriched 420-fold in pluripotent stemcells capable of initiating long-term bone marrow cultures (LTBMC) overunmanipulated bone marrow mononucleocytes (BMMNC) obtained afterFicoll-Hypaque separation, (Verfaillie et al., J. Exp. Med. 172, 509,1990). Positive selection for small blast-like cells that are CD34antigen positive but HLA-DR antigen negative was combined with a moreextensive negative selection to deplete the population of CD2, CD19 andCD71-positive cells.

The isolation process can initially use a “relatively crude” separationto remove major cell families from the bone marrow or otherhematopoietic cell source. If desired, large numbers of cells, namelymajor cell populations of the hematopoietic system such as T-cells,various lineages, such as B-cells, both pre-B and B-cells, granulocytes,myelomonocytic cells, and platelets, or minor cell populations, such asmegakaryocytes, mast cells, eosinophils and basophils can be removedusing initially magnetic bead separations. Optionally, in certainpopulations of progenitor cells, at least about 70%, usually 80% or moreof the total hematopoietic cells can be removed using conventionalmethods. It is not essential to remove every dedicated cell class,particularly the minor population members, and the platelets anderythrocytes, at the initial stage.

The separation techniques employed should maximize the retention ofviability of the fraction to be collected. For “relatively crude”separations, that is, separations where up to 10%, usually not more thanabout 5%, preferably not more than about 1%, of the total cells presenthaving a selected marker, may remain with the cell population to beretained, various techniques of differing efficacy may be employed. Theparticular technique employed will depend upon efficiency of separation,cytotoxicity of the methodology, ease and speed of performance, andnecessity for sophisticated equipment and/or technical skill. Proceduresfor separation may include magnetic separation, using antibody-coatedmagnetic beads, affinity chromatography, cytotoxic agents joined to amonoclonal antibody or used in conjunction with a monoclonal antibody,e.g. complement and cytotoxins, and “panning” with antibody attached toa solid matrix, e.g. plate. Techniques providing accurate separationinclude fluorescence activated cell sorters, which can have varyingdegrees of sophistication, e.g. a plurality of color channels, low angleand obtuse light scattering detecting channels, impedance channels, etc.

As exemplary of the subject method, in a first stage after incubatingthe cells from the bone marrow for a short period of time at reducedtemperatures, generally −10° to 10° C., with saturating levels ofantibodies specific for T-cell determinants, the cells are washed with afetal calf serum (FCS) cushion. The washed cells are then suspended in abuffer medium as described above and separated by means of theantibodies for the T-cell determinants.

Conveniently, the antibodies may be conjugated with markers, such asmagnetic beads, which allow for direct separation, biotin, which can beremoved with avidin bound to a support, fluorescers, e.g. fluorescein,which can use a fluorescence activated cell sorter, or the like, toallow for ease of separation of the T-cells from the other cells. Anytechnique may be employed which is not detrimental to the viability ofthe remaining cells.

Once the cells bound to the antibodies are removed, they may then bediscarded. The remaining cells may then be incubated for a sufficienttime at reduced temperature with a saturating level of antibodiesspecific for one or a mixture of cell differentiation antigens. The sameor different mechanism for selecting for these cells as was used forremoving the T-cells may be employed, where in the subject step, it isintended to use the unbound cells in subsequent stages.

The cells selected for as having the cell differentiation antigen arethen treated successively or in a single stage with antibodies specificfor the B-cell lineage, myelomonocytic, lineage, the granulocyticlineage, the megakaryocytic lineage, platelets, erythocytes, etc.,although minor lineages may be retained, to be removed later. The cellsbinding to these antibodies are removed as described above, withresidual cells desirably collected in a medium comprising fetal calfserum.

The residual cells are then treated with labeled antibodies selectivebut not specific for the stem cells, for mice the antibodies Sca-1 andThy-1^(lo), where the labels desirably provide for fluorescenceactivated cell separation (FACS). Multi-color analysis may be employedat this stage or previously. The cells are separated on the basis of anintermediate level of staining for the cell differentiation antigen, ahigh level of staining for Sca-1 and selected against dead cells andT-cells by providing for dyes associated with dead cells and T-cells asagainst stem cells. Other techniques for positive selection may beemployed, which permit accurate separation, such as affinity columns,and the like. The method should permit the removal to a residual amountof less than about 1% of the non-stem or non-progenitor cellpopulations.

The particular order of separation is not critical to this invention,but the order indicated is preferred. Preferably, cells will beinitially separated by markers indicating unwanted cells, negativeselection, followed by separations for markers or marker levelsindicating the cells belong to the stem cell population, positiveselection.

Compositions having greater than 90%, usually greater than about 95%, ofhematopoietic stem or progenitor cells may be achieved in this manner.Stem cells can be identified for example by having a low level of theThy-1 cell differentiation antigen, being negative for the variouslineage associated antigens and being positive for the Sca-1 antigen,which Sca-1 antigen is associated with clonogenic bone marrow precursorsof thymocytes and progeny T-cells, or as already indicated, the humancounterparts thereof.

However, the hematopoietic stem and progenitor cells that can be usedaccording to the invention are not limited to cells expressing theaforementioned cell surface molecules. Any suitable assay fordetermining the capacity of cells as hematopoietic progenitor or stemcells can be used. In a first example, the stem cell activity of a humancandidate cell is examined in vitro by testing its colony formingpotential (McAdams, 1996, TIBTECH 12:341-349 and Ploemacher et al.,Blood 79:834-837 (1992), the disclosures of which are incorporatedherein by reference).

In preferred embodiments, hematopoietic stem cells according to theinvention are characterized as having the ability to fully reconstitutethe bone marrow of a lethally irradiated host. One assay for stem cellactivity is an in vivo long-term marrow repopulating assay (MRA). Forthe assessment of mouse MRA, lethally irradiated mice can betransplanted with a bone marrow suspension and sacrificed 13 days aftertransplantation. The femoral cell content of the sacrificed mice istransplanted into secondary recipients and subsequently analyzed forcolony forming units (CFU-S-12, CFU-GM capacity) (Ploemacher and Brons,Exp. Hematol. 16:21-26, 1988 and Hematol., 16:27-32, 1998, thedisclosures of which are incorporated herein by reference). Cellsgenerating colonies in secondary recipients are deemed to be derivedfrom marrow seeded by stem cells in primary recipients. Anotherpreferred assay is the long-term repopulating assay (LTRA) whichidentifies hematopoietic stem cell by allowing measurements ofrepopulating activity over longer times than the MRA wherein mice aresacrificed at 13 days (Jones et al, 1990, Nature 347:188-189; Li et al.,1992, J. Exp. Med 175:1443-1447; Morrison and Weissman, 1994, Immunity1:661-673; Spangrude, 1995, Blood 85:1006-1016, the disclosures of whichare incorporated herein by reference).

Animal models for the measurement of human MRA have also been developed,including a sheep in utero transplantation system in which humanhematopoietic progenitor cells are transferred into the sheep fetusbefore the development of the ovine immune system. The presence ofabsence of human blood cells is then followed after the sheep is born.In another example, an assays test the ability of a candidate stem cellto repopulate the bone marrow of sublethally irradiated immune-deficientnon-obese diabetic/SCID (NOD/SCID) mice. (See Lapidot T., et al.,Science 255:1137, 1992; Vormoor et al., Blood 83:2489, 1994; Larochelleet al., Hum. Mol. Genet93. 4:163, 1995; Larochelle et al., Nat. Med.2:1329, 1996, the disclosures of which are incorporated herein byreference).

Once progenitor or stem cells have been isolated, they may be propagatedby growing in any suitable medium. For example, progenitor or stem cellscan be grown in conditioned medium from stromal cells, such as thosethat can be obtained from bone marrow or liver associated with thesecretion of factors, or in medium comprising cell surface factorssupporting the proliferation of stem cells. Stromal cells may be freedof hematopoietic cells employing appropriate monoclonal antibodies forremoval of the undesired cells, for example, with antibody-toxinconjugates, antibody and complement, etc.

Modifying Cells

The hematopoietic stem cells may be genetically modified by introducinggenetic material into the cells, for example using recombinantexpression vectors.

A recombinant expression vector preferably comprises an assembly of (1)a genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription initiation and terminationsequences. Structural units intended for use in eukaryotic expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it may include an N-terminal methionine residue. This residue may or maynot be subsequently cleaved from the expressed recombinant protein toprovide a final product.

Examples of suitable promoters which may be employed include, but arenot limited to, TRAP promoter, adenoviral promoters, such as theadenoviral major late promoter; the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; the Rous Sarcoma promoter;inducible promoters, such as the MMT promoter, the metallotlioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; ITRs;the β-actic promoter; and human growth hormone promoters. Preferably thepromoter will be capable of driving expression of a gene operably linkedthereto in a hematopoeitic cell; in one example the elongation factor 1α(EF 1α) promoter is used, which allows homogeneous expression in allhematopoietic cell types and particularly in NOD-SCID repopulating cells(Sirven, A. et al., Mol. Ther. 3, 438-448, 2001, the disclosure of whichis incorporated herein by reference). The promoter also may be thenative promoter that controls the gene encoding the polypeptide. Thesevectors also make it possible to regulate the production of thepolypeptide by the engineered progenitor cells. The selection of asuitable promoter will be apparent to those skilled in the art.

The human hematopoietic stem cells thus may have stably integrated arecombinant transcriptional unit into chromosomal DNA or carry therecombinant transcriptional unit as a component of a resident plasmid.Cells may be engineered with a polynucleotide (DNA or RNA) encoding apolypeptide ex vivo, for example. Cells may be engineered by proceduresknown in the art by use of a retroviral particle containing RNA encodinga polypeptide.

Various methods are available for genetically modifying donor cellsprior to implantation into a recipient subject. Suhr, S. T. and Gage, F.H., 1993, Arch. Neurol. 50(11):1252-1268; Gage, F. H. et al., 1987,Neuroscience 23(3):795-807. These methods include direct DNA uptake(transfection), and infection with viral vectors such as lentivirus,retrovirus, herpes virus, adenovirus, and adeno-associated virusvectors. Suhr, S. T. et al., 1993, Arch. Neurol. 50:1252-1268.Transfection can be effected by endocytosis of precipitated DNA, fusionof liposomes containing DNA or electroporation. Suhr, S. T. et al.,1993, Arch. Neurol. 50:1252-1268. Another method of transfecting donorcells is through the use of a “gene gun”. In this method, microscopicDNA-coated particles are accelerated at high speeds through a focusingtube and “shot” or injected into cells in vitro (Klein, R. M. et al.,1992, Biotechnology 24:384-386; Zelenin, A. V. et al., 1989, FEBS Lett.,244:65-67) or in vivo (Zelenin, A. V. et al., 1991, FEBS Lett.,280:94-96). The cells close around the wound site and express genescarried into the cell on the particles. All of the above-referenced areincorporated herein by reference.

Retroviral vectors typically offer the most efficient and bestcharacterized means of introducing and expressing foreign genes incells, particularly mammalian cells, These vectors have very broad hostand cell type ranges, integrate by reasonably well understood mechanismsinto random sites in the host genome, express genes stably andefficiently, and under most conditions do not kill or obviously damagetheir host cells. The methods of preparation of retroviral vectors havebeen reviewed extensively in the literature (Suhr, S. T. and Gage, F.H., 1993, Arch. Neurol. 50(11):1252-1258; Ray, J. and Gage, F. H., 1992,Biotechniques 13(4):598-603; Anderson, W. F., 1984, Science 226:401-409;Constantini, F. et al., 1986 Science 233:1192-1194; Gilboa, E. et al.,1986, Biotechniques 4:504-512; Mann, R. et al., 1983, Cell 33:153-159;Miller, A. D. et al., 1985, Mol. Cell Biol. 5:431-437; and Readhead, C.et al., 1987, Cell 48:703-712) and are now in common use in manylaboratories. Suitable vectors and improved methods for production ofrecombinant retroviral vectors are also provided in U.S. Pat. No6,013,516. Other techniques for producing genetically modified cells aredescribed in detail in PCT publication WO 95/27042. All of theabove-referenced are incorporated herein by reference.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus (e.g. HIV-1), adenovirus,Myeloproliferative Sarcoma Virus; and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is MGIN, derived from murineembryonic stem cells.

In preferred embodiments, lentiviral vectors are used due to theirability to introduce genes into non-dividing or post-mitotic cells.Lentiviral vectors also do not suffer from low viral titer limitationsas do certain other vectors. Lentiviruses are complex retroviruses,which, in addition to the common retroviral genes gag, pol and env,contain other genes with regulator or structural function. The highercomplexity enables the virus to regulate its life cycle, as in thecourse of latent infection. A typical lentivirus is HumanImmunodeficiency Virus (HIV), the etiologic agent of AIDS. In vivo, HIVcan infect macrophages which are terminally differentiated cells thatrarely divide. In vitro, HIV can infect primary cultures ofmonocyte-derived macrophages (MDM), and HeLA-Cd4 or T-lymphoid cellsarrested in the cell cycle by treatment with aphidicolin or gammairradiation. Infection of these cells is dependent on the active nuclearimport of HIV preintegration complexes through the nuclear pores of thetarget cells. This occurs by the interaction of multiple, partlyredundant, molecular determinants in the complex with the nuclear importmachinery of the target cell. Identified determinants include afunctional nuclear localization signal (NLS) in the gag MA protein, thekaryophilic virion-associated protein vpr, and a C-terminalphosphotyrosine residue in the subset of the gag MA protein.

The recently described HIV based lentiviral vector has been shown to beefficient in integrating into non-cycling cells (Verma, Nature389:239-242, 1997). Studies to determine the usefulness of this vectorhave been performed by Choi and Gewirtz (1998, Blood 92:468a). To obtainbetter expression, Uchida et al. (1998, PNAS USA 95:11939-11944)successfully utilized a HIV-based vector system that also expressed theviral transcription co-factor tat that is critical for high expressionof the HIV LTR. A hybrid HIV/murine stem cell virus (NSCV) vector hasalso been developed where in the original internal CMS enhancer/promoteris removed and the U3 region of the HIV LTR is partially replaced by theU3 region of the MSCV LTR for increased safety with a high transductionefficiency (U.S. Pat. No. 6,218,186). All of the above-referenced areincorporated herein by reference.

A lentivirus vector may be an attenuated virus that has been modified sothat it is incapable of causing disease of pathology in a host animal orcell (i.e. it encompasses virus that are incapable of causing or causereduced cytopathic effects in viral cultures). Viral particles may becapable of some degree of infection and gene expression, but are notable to produce disease or productive infection.

Vectors for gene transfer into hematopoietic cells are also reviewed inElwood, Leuk. Lymphoma 2001, 41 (5-6):465-482, incorporated herein byreference.

It is also possible to use vehicles other than retroviruses togenetically engineer or modify the hematopoietic stem cells. Geneticinformation of interest can be introduced by means of any virus whichcan express the new genetic material in such cells. For example, SV40,herpes virus, adenovirus, adeno-associated virus and humanpapillomavirus can be used for this purpose. Other methods can also beused for introducing cloned eukaryotic DNAs into cultured manmmaliancells, for example, the genetic material to be transferred to stem cellsmay be in the form of viral nucleic acids.

In addition, the expression vectors may contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedcells such as dihydrofolate reductase or neomycin resistance.

The hematopoietic cells may be transfected through other means known inthe art. Such means include, but are not limited to transfectionmediated by calcium phosphate or DEAE-dextran; transfection mediated bythe polycation Polybrene; protoplast fusion; electroporation; liposomes,either through encapsulation of DNA or RNA within liposomes, followed byfusion of the liposomes with the cell membrane or, DNA coated with asynthetic cationic lipid can be introduced into cells by fusion.

The present invention further makes it possible to genetically engineerhuman hematopoietic stem or progenitor cells in such a manner that theyproduce polypeptides, hormones and proteins not normally produced inhuman hematopoietic cells or in microglia or other cells of the CNS inbiologically significant amounts or produced in small amounts but insituations in which regulatory expression would lead to a therapeuticbenefit. For example, the hematopoietic stem cells could be engineeredwith a gene that expresses a molecule that specifically inhibitsneurodegeneration. Alternatively the cells could be modified such that aprotein normally expressed will be expressed at much lower levels. Theseproducts would then be secreted into the surrounding media or purifiedfrom the cells. The human hematopoietic stem cells formed in this waycan serve as continuous short term or long term production systems ofthe expressed substance. These genes can express, for example, hormones,growth factors, matrix proteins, cell membrane proteins, cytokines,adhesion molecules, “rebuilding” proteins important in tissue repair.The expression of the exogenous genetic material in vivo is oftenreferred to as “gene therapy”.

Nucleic Acids

As will be appreciated by the skilled person, according to theinvention, the cells may be engineered to express any suitable nucleicacid sequence. In one aspect, a nucleic acid sequence may serve toexpress a nucleic acid acting directly on a biological target, such asin an antisense or ribozyme treatment. In other aspects, said nucleicacid sequence may encode a polypeptide. As used herein, the termspeptide and polypeptides are used interchangeably, as polypeptides ofessentially any length may be used in accordance with the presentinvention. Polypeptides may be full-length polypeptides or fragmentsthereof suitable for a particular application (e.g. capable of restoringa biological activity, inhibiting a biological activity). Polypeptidesmay be secreted or non-secreted polypeptides.

A nucleic acid can encode a functionally active polypeptide or aninhibitor, e.g. of a target polypeptide or an inhibitor of a bindingevent. For example, a polypeptide may be a dominant negative mutantpolypeptide. Non-limiting examples of nucleic acids that can beexpressed include nucleic acids encoding neuropeptides,neurotransmitters, enzymes involved in biosynthesis, proteins involvedin intracellular signalling pathways, antibodies, pro- oranti-inflammatory molecules (for example cytokines), and receptors. Forexample, viral vectors have been developed encoding enzymes responsiblefor doparine biosynthesis (Freese et al., 1997, Epilepsia 38(7):759-766) and the GluR6 excitatory amino acid receptor subtype(Bergold et al., 1993, PNAS USA 90: 6165-6169). In certain applications,nucleic acids may allow detection of virions and/or detection oftransgene expression. Nucleic acids may encode detectable markerpolypeptides, such as a fluorescent protein (ex. GFP) or anotherdetectable polypeptide such as β-galactosidase. Other non-limitingexamples of genes suitable for use according to the invention includeanti-apoptotic genes such as bcl-2, interleukin-1 converting enzyme,crmA, bcl-xl, FLIP, survivin, IAP, ILP; genes which provides targetcells, preferably tumor cells, with enhanced susceptibility to aselected cytotoxic agent, such as the herpes simplex virus thymidinekinase (HSV-tk), cytochrome P450, human deoxycytidine kinase, andbacterial cytosine deaminase genes (see also Springer andNiculescu-Duvaz, 2000, J. Clin. Invest., 105:1161-1167). Also includedare polypeptides which reduce glutamate toxicity, and polypeptides withact as calcium buffers or binding protein such as calbindin. Alsoencompassed are polypeptides capable of inhibiting the activity of anenzyme. For example, encompassed in Alzheimer's disease are apolypeptide capable of inhibiting or reducing the formation of Aβproduction, a polypeptide capable of modifying APP processing, apolypeptide capable of stimulating or generally increasing α-secretasecleavage activity, a polypeptide capable of inhibiting the β-secretasepathway, a polypeptide capable of inhibiting the γ-secretase pathway, ora polypeptide capable of inhibiting tau protein hyperphosphorylation.

Other examples of nucleic acids that can be used with the presentinvention include nucleic acids coding for growth factors orneurotrophic factors, including but not limited to genes encoding:acidic fibroblast growth factor (aFGF; FGF-1); glial cell line-derivedneurotrophic factor; brain-derived neurotrophic factor; nerve growthfactor; TGF-α, extracellular matrix proteins (collagens, fibronectins,integrins); ornithine amino transferase; prostaglandin synthesisregulation proteins; trabecular meshwork proteins; NT-3, NT-4/5;hypoxanthine phosphoribosyltransferase; tyrosine hydroxylase,prostaglandin receptors, catalase and glutathione peroxidase; sequencesencoding interferons, lymphokines, cytokines (cytokines acting in ananti-inflammatory manner such as TGF-β, IL-4, IL-10 or IL-13,proinflammatory cytokines such as IL-6) and antagonists thereof such astumor necrosis factor (TNF), CD4 specific antibodies, and TNF or CD4receptors; sequences encoding the GABA synthesizing enzyme glutamic aciddecarboxylase (GAD), calcium dependent potassium channels orATP-sensitive potassium channels; and sequences encoding thymidinekinase. Also envisioned are sequences encoding antisense nucleic acids.Other examples of polypeptides that can be encoded includedopadecarboxylase, cell adhesion molecules, interleukin-1β, superoxidedismutase, basic fibroblast growth factor, ciliary neurotroplhic factorand neurotransmitter receptors.

Nucleotide sequences encoding these polypeptides are known to those ofskill in the art. For example, Abraham et al., Science 233:545, 1986,disclose the nucleotide sequence of bovine bFGF, while the-nucleotidesequence of human bFGF is disclosed by Abraham et al., EMBO J., 5:2523,1986. Mergia et al., Biochem. Biophys. Res. Conmmun. 164:1121, 1989,provide the nucleotide sequence of the human aFGF gene. The nucleotidesequence of the rat glial cell line-derived neurotrophic factor isdescribed by Springer et al., Exp. Neurol., 131:47, 1995. Maisonpierreet al., Genomics 10:558, 1991, provide the nucleotide sequences of humanand rat brain-derived neurotrophic factor, while Arab et al., Gene185:95, 1997, disclose the amino acid sequence of bovine brain-derivedneurotrophic factor. Rat ciliary neurotrophic factor is described byStocki et al., Nature 342:920, 1989. The nucleotide sequence of thehuman ciliary neurotrophic factor gene is disclosed by Negro et al.,Eur. J. Biochem., 201:289 1991, Lin et al., Science, 246:1023, 1989, andby Lam et al., Gene, 102:271, 1991. Ulrich et al., Nature, 303:821,1983, provide a comparison of human and murine coding regions ofbeta-nerve growth factor genes. The nucleotide sequence of bovineinterleukin-1β is disclosed by Leong et al., Nucl. Acids Res., 16:9054,1988, while Bensi et al., Gene, 52:95, 1987, provide the nucleotidesequence of the human interleukin-1β gene. All of the above-referencedare incorporated herein by reference.

DNA molecules encoding such polypeptides can be obtained by screeningcDNA or genomic libraries with polynucleotide probes having nucleotidesequences based upon known genes. Standard methods are well-known tothose of skill in the art. See, for example, Ausubel et al. (eds.),SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages 2-1 to 2-13 and5-1 to 5-6 (John Wiley & Sons, Inc. 1995).

Alternatively, DNA molecules encoding growth factors can be obtained bysynthesizing DNA molecules using mutually priming long oligonucleotides.See, for example, Ausubel et al., pages 8.2.8 to 8.2.13 snf pages 8-8 to8-9. Also, see Wosnick et al., Gene, 60:115, 1987. Establishedtechniques using the polymerase chain reaction provide the ability tosynthesize DNA molecules at least two kilobases in length. Adang et al.,Plant Molec. Biol., 21:1131, 1993; Bambot et al., PCR Methods andApplications 2:266, 1993; Dillon et al., “Use of the Polymerase ChainReaction for the Rapid Construction of Synthetic Genes,” in METHODS INMOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS ANDAPPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc., 1993);Holowachuk et al., PCR Methods Appl., 4:299, 1995).

Preparation of Cells

Pharmaceutical Compositions

The cells of the invention can be inserted into a delivery device whichfacilitates introduction by e.g., injection, of the cells into thesubjects. Such delivery devices include tubes; e.g., catheters, forinjecting cells and fluids into the body of a recipient subject. In apreferred embodiment, the tubes additionally have a needle, e.g., asyringe, through which the cells of the invention can be introduced intothe subject at a desired location. The hematopoietic progenitor cells ofthe invention can be inserted into such a delivery device, e.g., asyringe, in the form of a solution.

Carriers for these cells can include but are not limited to solutions ofphosphate buffered saline (PBS) containing a mixture of salts inphysiologic concentrations. As used herein, the term “solution” includesa pharmaceutically acceptable carrier or diluent in which the cells ofthe invention remain viable. Pharmaceutically acceptable carriers anddiluents include saline, aqueous buffer solutions, solvents and/ordispersion media. The use of such carriers and diluents is well known inthe art. The solution is preferably sterile and fluid to the extent thateasy syringability exists. Preferably, the solution is stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi thoughthe use of, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. Solutions of the invention can be prepared byincorporating cells as described herein in a pharmaceutically acceptablecarrier or diluent and, as required, other ingredients enumerated above,followed by filtered sterilization.

Cell Culture

Compositions enriched in hematopoietic stem or progenitor cellsaccording to the invention can be maintained or expanded in cultureprior to administration to a subject. Culture conditions are generallyknown in the art depending on the cell type. Conditions for themaintenance of CD34+ in particular have been well studied, and severalsuitable methods are available.

A common approach to ex vivo multi-potential hematopoietic cellexpansion is to culture purified progenitor or stem cells in thepresence of early-acting cytokines such as interleukin-3. It has alsobeen shown that inclusion, in a nutritive medium for maintaininghematopoietic progenitor cells ex vivo, of a combination ofthrombopoietin (TPO), stem cell factor (SCE), and flt3 ligand (Flt-3L;i.e., the ligand of the flt3 gene product) was useful for expandingprimitive (i.e., relatively non-differentiated) human hematopoieticprogenitor cells in vitro, and that those cells were capable ofengraftment in SCID-hu mice (Luens et al., 1998, Blood 91:1206-1215). Inother known methods, cells can be maintained in vitro in a nutritivemedium (e.g., for minutes, hours, or 3, 6, 9, 13, or more days)comprising murine prolactin-like protein E (mPLP-E) or murineprolactin-like protein F (mPIP-F; collectively mPLP-E/IF) (U.S. Pat. No.6,261,841). It will be appreciated that other suitable cell culture andexpansion method can be used in accordance with the invention as well.Cells can also be grown in serum-free medium, described in U.S. Pat. No.5,945,337. All of the above-referenced are incorporated herein byreference.

Cell Compositions

The invention also relates to isolated hematopoietic progenitor or stemcells as described herein, and to con positions of cells enriched inherliatopoietic progenitor or stem cells capable of migrating to the CNSof a subject, and/or capable or giving rise to microglia and/or capableof expressing a therapeutic polypeptide of interest in the CNS of asubject. Said hematopoietic progenitor or stem cells will give rise tomicroglia in the brain of a subject following administration by asuitable method, preferably administration outside of the CNS such asfor example intravenous administration.

The invention encompasses hematopoietic progenitor or stem cellcompositions specifically adapted for expressing a protein in the CNS ofa mammalian subject. Said cell compositions include a huitanhematopoietic progenitor or stem cell comprising an expression vector,preferably transduced with a lentiviral vector, comprising an assemblyof (1) a genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers allowing expression of atherapeutic gene operably linked thereto when the cell is present in theCNS of a subject, (2) a structural or coding sequence which istranscribed into mRNA and translated into a therapeutic polypeptide, and(3) appropriate transcription initiation and termination sequences. Atherapeutic polypeptide may be a polypeptide normally expressed in thehuman CNS. Other examples include polypeptides capable of stimulating orencouraging the growth of CNS cells (e.g. neurons, glial cells) andpolypeptides capable of inhibiting neurodegeneration. Preferably thecell composition is capable of stably expressing a therapeuticpolypeptide in the CNS of a mammal. Preferably the transduced cellprovides an individual with a CNS disease with a biologically activetherapeutic molecule in an amount sufficient to anheliorate a symptom orfeature of the CNS disease.

Administration

Delivery of the transduced cells according to the invention may beeffected using various methods and includes most preferably intravenousadministration by infusion as well as direct depot injection intoperiosteal, bone marrow and/or subcutaneous sites.

Upon administration, the cells will generally require a period of timeto engraft. It is generally preferable to have the highest percentage ofengraftment possible, preferred embodiments comprises achievingengraftment of at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, orsubstantially all of the cells in the bone marrow of a subject.Achieving a high level of engraftment of hematopoietic stem orprogenitor cells typically takes a period week to months.

Generally, the recipient will be treated to enhance engraftment, using aradiation or chemotheraptic treatment prior to the administration of thecells.

In general, hematopoietic progenitor or stemr cells to be administeredto a subject will be autologous, e.g. derived from the subject.Nevertheless, allogeneic hematopoietic cell transplants are alsoenvisioned, and allogeneic bone marrow transplants are carried outroutinely. Allogeneic cell transplantation can be offered to thosepatients who lack an appropriate sibling donor by using bone marrow fromantigenically matched, genetically unrelated donors (identified througha national registry), or by using hematopoietic progenitor or stem-cellsobtained or derived from a genetically related sibling or parent whosetransplantation antigens differ by one to three of six human leukocyteantigens from those of the patient.

Treatment

The cells and methods of this invention will be useful as providing ameans for delivering a desired biologically active molecule to the CNS,e.g. to protect the endogenous affected host tissue against variousneurodegenerative processes. Preferably the molecule is a secretedprotein. In this aspect, a disorder relating to substantially any CNScell population can be treated.

In another aspect, the invention can be useful in the treatment of adisorder affecting, caused by or mediated by microglia. It iscontemplated that the cells can replace diseased, damaged or lostmicroglia in the host. Alternatively, the transplanted issue may augmentthe function of the endogenous affected host microglia.

As described further herein, the transplanted cells may also begenetically modified to provide a biologically active molecule that istherapeutically effective. These cells may find use in the treatment ofCNS disorders, including for example metabolic disorders such as obesityhaving a basis in the CNS.

Thus, in one aspect, an exogenously administered active factor isprovided, e.g. for providing or augmenting a function in a subject,including for the treatment of an active factor deficiency disorder and,in particular, the treatment of diseases and disorders which may beremedied by treatment with active factors, such as neurotransmitters,neuromodulators, hormones, trophic factors, cofactors, and growthfactors. All these substances are characterized by the fact they aresecreted by “source” cells and produce a specific change in a “target”cell or in the source cell itself. Any suitable active factor can beprovided, including any of the examples provided in the section hereintitled “nucleic acids”.

Deficits in active factors have been implicated in disease with verydifferent phenotypes. For example, lack of neurotransmitter-mediatedsynaptic contact causes neuropathological symptoms, and can also lead tothe ultimate destruction of the neurons involved.

According to the present invention, hematopoietic progenitor or stemcells which give rise to CNS cells, particularly microglia, may serve tosecrete a diffusible gene product that can be taken up and used bynearby target cells. One strategy that has been pursued in animal modelsof neurodegenerative disease is to augment neurotransmitter functionwithin the brain through tissue transplantation. This may proveparticularly advantageous for the treatment of disorders in whichdiffuse delivery across the brain is required, such as in the case ofAlzheimer's disease.

Two non-limiting examples of potential therapeutic uses of engineeredmicroglia through the transplantation of genetically manipulated humanCD34+ cells are further described as follows for the treatment of HIVdementia complex and Alzheimer's disease.

Manipulating Microglia in HIV Dementia Complex

Monocytes play a role in the entry of HIV into the CNS, in viralpersistence in the CNS and in mediating neuronal injury. Thetransplantation of autologous genetically modified CD34+ cells offersthe possibility to replace endogenous microglia by new microglial cellsthat would express mutated form of CCR5 receptors, allowing these cellsto become resistant to HIV infection within the brain. Alternatively,microglia can be genetically modified to inhibit the secretion of TNFαthat occurs after binding of gp120 on their CXCR4 receptors. This couldbe achieved by expressing mutated form of CXCR4 at the surface ofmicroglia. Microglia can also be modified in order they express anantagonist ligand of the CXCR4 receptor or a factor that inhibitdownstream signaling from CXCR4 receptor (Davis et al., J. Exp. Med.,186:1793-1793, 1997 incorporated herein by reference).

Manipulating Microglia in Alzheimer's Disease

The presence of microglia in senile plaques offers a number of targetsfor therapeutic intervention. Most of them could be achieved through thereplacement of endogenous microglia by new microglia afterautotransplantation of genetically modified CD34+ cells. These targetsinclude: 1) the signaling steps that lead to neuronal damage becausemicroglia are activated in the presence of Aβ-containing plaques; 2) theup-regulation of Aβ clearance by nucroglia; 3) the interruption of Aβbinding to microglia; 4) the production of survival neuronal factors.

In one aspect the invention involves a method of treatment comprisingthe interruption of a signaling inflammatory cascade that leads toneuronal damage by providing a hematopoietic stem or progenitor cellcapable of giving rise to microglia. Microglia can be provided whoseexpression of C1 inhibitor is up-regulated allowing the inactivation ofcomplement pathway, or microglia can be provided that express aninhibitor of COX-2 activity. A number of retrospective clinicalobservations, as well as epidemiological data, have suggested thatanti-inflammatory drugs may offer protection against AD.

In one aspect the invention involves a method of treatment comprisingup-regulation of Aβ processing by providing a hematopoietic stem orprogenitor cell capable of giving rise to microglia. Microglia can bemade to overexpress the cytokine TGF-β1 (Wyss-Coray T. et al., Nat.Medecine, 7:612-618, 2001). TGF-β1 may directly stimulate microglia tophagocytose Aβ peptides or alternatively induce the secretion ofAβ-binding proteins by astrocytes, which facilitate microglialphagocytosis.

In another aspect, the invention involves a method of treatmentcomprising preventing Aβ binding to microglia by providing ahematopoietic stem or progenitor cell capable of giving rise tomicroglia. Aβ binding to microglia may activate microglia and henceleads to neuronal damage. This process can be inhibited by providingmicroglia that would secrete HHQK-like peptides that, in turn, willimpede the binding of Aβ peptides to microglia type-A macrophagescavenger receptor. This strategy offers the advantage to suppress onlythe toxicity that occurs during Aβ-dependent activation of microgliawithout impairing their other immune functions.

In other strategies, microglia can be genetically modified to expressneuronal trophic factors. NGF is promising given it protects cholinergicneurons from axotomy-induced cell death in fimbria-fornix lesion models,reverses age-associated atrophy of cholinergic cell bodies and improvesspatial navigation, memory and learning in mice.

Manipulation Neuronal NF-κB Activation in Microglia to IncreaseNeuroprotection

Based on work in animal models, manipulation of NF-κB signaling may bevaluable in treating several neurodegenerative disorders, includingAlzheimer's disease (AD) and Parkinson's disease (PD) (Mattson M. P. andCamandola S. J., Clin. Invest., 107:247-254, 2001).

Functional NF-κB complexes (p50, P65 and IκBα) are present in microgliaand neurons. NF-κB influences the expression of a complex array of genesin the CNS, and in general, theses genes serve important functions incellular responses to injury. NF-κB is activated by signals thatactivate IκB kinase (IKK), resulting in phosphorylation of IκBα. Thistargets IκBα for degradation in the proteosome and frees p65-p50 dimer,which then translocates to the nucleus and binds to consensus κBsequences in the enhancer region of κB-responsive genes. In general, itappears that genes activated by NF-κB in neurons protect them againstdegeneration whereas activation of NF-κB in macroglia promotes neuronaldegeneration.

In AD, TNFα can protect neurons against Aβ-induced death via a NF-κBmediated mechanism. α-secretase-derived form of secreted amyloidprecursor protein (sAPPα is potently excitoprotective and antiapopoticin CNS neurons. NF-κB activation following exposure to sAPPα iscorrelated with increased resistance of neurons to metabolic andexcitotoxic insults. As a result of aberrant proteolytic processing ofβAPP, levels of sAPPα may be decreased. It seems likely that activationof NF-κB in neurons associated with amyloid deposit is a cytoprotectiveresponse. On the other hand, the increased levels of membrane lipidperoxidation that occur in neurons degenerating in AD may endangerneurons by suppressing NF-κB activation. This is the case of4-hydroxynonenal which inhibits NF-κB activation. Moreover, prostateapoptosis response-4 (Par-4), a proapoptotic protein implicated in thepathogenesis of neuronal degeneration in AD, strongly suppresses NF-κBactivation in cultured neural cells.

Immunohistochemical analyses of brain sections from PD patients show a70 fold increase of nuclear p65 NF-κB protein in dopaminergic neurons ofsubstantia nigria. Spinal cords of patients with amyotrophic lateralsclerosis show increased NF-κB activation in astrocytes associated withdegenerating motor neurons. In both diseases, the increased NF-κBactivity in the affected neurons may represent an early protectiveresponse to ongoing oxidative stress and mitochondrial dysfunction.

Exitotoxic and ischemic injury to neurons is mediated in part bydysregulation of cellular calcium homeostasis resulting in a prolongedelevation of intracellular calcium levels. Activation of NF-κB inneurons can stabilize intracellular calcium levels under ischemia-likeconditions. This may result from induction of several different genes,including those encoding calcium-binding proteins (like calbindin) andglutamate receptor subunits.

Although activation of NF-κB in neurons can prevent apoptosis in thesecells, NF-κB activation in microglia may indirectly lead to apoptosis ofother cells by promoting production of cytotoxic agents such as nitricoxide. Cytokine-mediated activation of microglia may explain the abilityof inhibitors of NF-κB to protect against cell damage in certainexperimental paradigms that involve an inflammatory responses in whichmicroglia is activated. Microglial activation is associated with amarked increase in COX-2, an oxyradical-generating enzyme, and agentsthat inhibit NF-κB activation can suppress LPS (liposaccharide)-inducedCOX-2 expression.

The transplantation of autologous genetically modified CD34+ cellsoffers the possibility to replace endogenous microglia by new microglialcells that could activate NF-κB in neurons. On the other hand, it isalso possible to replace endogenous microglia by genetically modifiedmicroglia in which activation of NF-κB pathway leading to deleteriouseffects is inhibited.

In one approach, the invention encompasses activating NF-κB in neuronsby transplanting genetically modified CD34+ cells whosederived-microglia will secrete sAPPα or activity-dependent neurotrophicfactor (ADNF), either of which are good candidates to activate NF-κB inneurons.

In another strategy, genetically modified CD34+ cells that willdifferentiate into microglia secreting at a low and regulated levelheat-shock proteins can be transplanted. Neurons exposed to low level ofheat-shock proteins can be preconditioned through NF-κB activation.Neuronal preconditioning increases resistance of neurons to variousoxidative, metabolic and excitotoxic insults in experimental modelsrelevants to AD, PD and Huntington's disease.

In a further strategy for inactivating NF-κB pathway in microglia,genetically modified CD34+ cells can be transplanted, the cells givingrise to microglia expressing proteosome inhibitors (which inhibit NF-κBactivation by preventing degradation of IκBα), peptides oroligonucleotide inhibitors that block DNA-binding activity of p50/p65dimers on consensus κB sequences.

Manipulating Microglia to Express Neurotophic Factors in AD, PD andMultiple Slcerosis

Neurotrophic factors are secreted peptides that are of potential valuesin several neurodegenerative diseases, including AD and PD (Siegel andChauhan, 2000). These diffusible proteins act via retrograde signalingpromoting neuronal surviving. For most of them, their systemic injectionlead to serious side effects that limit their clinical use. Onepossibility to circumvent these limitations would be to transplantgenetically CD34+ cells whose derived-microglia will secreteneurotrophic factors, likely in combination since many studies havedemonstrated that combined administration of neurotropic factors isoften synergistic. In multiple sclerosis, remyelinating “shadow” plaquescan be observed in the early acute phase of the disease but the rate ofremyelination is limited. One could envisage to transplant geneticallyCD34+ cells whose derived-microglia will secrete growth factors thatpromote differentiation of oligodendrocytes precursors and survival ofoligodendrocytes (reviewed in Diemel et al., 1998).

EXAMPLES Example 1 Transplantation of Human Modified CD34+ Cells canDifferentiate into Brain Microglia Expressing a Transgen. Materials andMethods

Lentiviral Vector

TRIP-ΔU3-EF1α-ALD lentiviral vector was constructed by replacing theenhanced green fluorescent protein (EGFP) cassette (BamHI/KpnI) from thepreviously described TRIP-ΔU3-EF1α-EGFP lentiviral vector (Sirven, A. etal., Blood 96, 4103-4110, 2000, and Sirven, A. et al., Mol. Ther., 3,438-448, 2001) by a BamHI-EcoRI fragment containing the coding sequenceof the human ALD cDNA (Mosser, J. et al., Nature, 361, 726-730, 1993).This self-inactivating (SIN) vector where the U3 region of the 3′LTR isdeleted to improve the safety of the vector system includes the centralpolypurine tract (cPPT) and the central termination sequence (CTS)(Zennou, V. et al., Cell, 101, 173-185, 2000) that increases the genetransduction efficiency in human CD34+ hematopoietic cells (Sirven etal., 2000). The expression of the ALD gene is driven by the elongationfactor 1α (EF 1α) promoter that allows homogeneous expression in allhematopoietic cell types and particularly in NOD-SCID repopulating cells(Sirven et al., 2001).

Preparation of High-Titer Virus Vector

Lentivirus vectors were generated by transient calcium phosphateco-transfection of 293T cells by the vector plasmid, an encapsidationplasmid lacking all accessory HIV-1 proteins (p8.91) and a VSV(vesicular stomatitis virus) envelope expression-plasmid (pHCMV-G), aspreviously described (Zennou et al., 2000).

Vector particles were normalized according to both p24 (HIV-A capsidprotein) content of supernatants (Zennou et al., 2000) and measurementof infectious titer on murine 3T3 cells (Cartier, N. et al., Proc. Natl.Acad. Sci. USA, 92, 1674-1678, 1995). Viral titers varied from 5.10⁸ to10⁹ IU/ml.

Isolation of ALD CD34⁺ Cells

CD34+ cells were isolated from granulocyte colony-stimulating factor(G-CSF)-mobilized peripheral blood from ALD patients according toapproved institutional guidelines. CD34⁺ cells were purified byimmuno-magnetic selection (Miltenyi Biotec, Paris, France) as previouslydescribed (Sirven, 2000 and 2001). Fluorescent activating cell sorting(FACS) analysis performed on a FACStar (Becton Dickinson) showed over90% purity of the CD34+ population. CD34⁺ cells were then stored inliquid nitrogen before use.

Transduction Protocol

CD34⁺ cells were plated at 10⁶ cells/ml in serum free medium (Stem CellTechnologies, Vanvouver, Canada) in the presence of 4 recombinant humancytokines: 10 ng/ml stem cell factor (SCF) (Amgen, Neuilly-sur-Seine,France); 10 ng/ml Flt3-Ligand (FL) (Immunex, Seattle, USA); 10 ng/mlinterleukin (IL)-3 (Novartis France, Rueil-Malmaison, France) and 10ng/ml pegylated-megacaryocyte-growth and differentiation factor(PEG-MGDF hereafter named TPO) (Kirin Brewery, Tokyo, Japan). Lentiviralvector particles were added twice at 0 and 12 hour at multiplicity ofinfection (MOI) of 5. At 36 hours, transduced and non-transduced CD34+cells were washed and cultured for 72 hours in H5100 long term culturemedium (StemCell Technology, Vancouver, Canada) on MS5 stromal cells.Expression of the human ALD protein (ALDP) was analyzed byimmunocytofluorescence (Cartier et al., 1995; Fouquet, F. et al.,Neurobiol. Dis., 3, 271-285, 1997; and Doerflinger N. et al., Hum. GeneTher., 9, 1025-1036, 1998).

Hematopoietic Cell Cultures

Colony forming cells (CFCs) and long-term culture-initiating cells(LTC-ICs) were assayed as described (Sirven et al., 2000 and 2001).

Bulk and 1/10/50 per well long-term culture (LTC) cells were studiedseparately. After 5 weeks, LTC cells were plated on methycelluloseplates and colonies were assessed 15 days later for ALDP expression.

Lymphoid (B, NK) and myeloid (granulo-monocytic) differentiation wasassessed on MS5 stronial cells in the presence of SCF, FL, TPO, IL-15and IL-2 as described (Sirven et al., 2000 and 2001).

Cells were phenotyped by FACS after 3-4 weeks of culture, using thefollowing mouse monoclonal antibodies (mAbs): CD19-PE (phycoerythrin)(Becton Dickinson) for B lymphocytes; CD15-PE and CD14-PE (PharMingen,Pont de Claix, France) for granulocytes and macrophages; CD56-PE-Cy5(Immunotech, Villepinte-Roissy CDG, France) for NK cells and CD34-PE-Cy5(Immunotech, Villepinte-Roissy CDG, France). Non-specific staining wasdetected using irrelevant mouse IgG1 and IgM mAbs.

Expression of ALDP was scored using immunocytochemistry with ananti-human ALDP antibody (Fouquet et al., 1997 and Doerflinger et al.,1998) in CFCs, LTC cells and cells from LTC giving rise to CFCs(LTC-Ics).

Expression of ALDP in monocytes-macrophages derived from lympho-myeloidcultures and LTC cells was analysed with polyclonal anti-human ALDP(Fouquet et al., 1997 and Doerflinger et al., 1998) and monoclonalanti-CD68 KP1 (Dako, Carpinteria, CA) antibodies after incubation withhorse anti-mouse IgG (H+L) antibody directly conjugated to fluorescein(Vector Laboratories) and biotinylated anti-rabbit IgG antibody andfurther incubation with Cy3-conjugated streptavidine (Chemicon).

Transplantation of Transduced ALD CD34⁺ Cells into NOD/SCID Mice

Immediately after transduction, 1.5-10⁶ ALD CD34⁺ cells wereintravenously injected into sub-lethally irradiated NOD-LtSz-scid/scidNOD/SCID) mice (3 Gy, at 0.43 Gy/mn; in a X-ray Phillips RT250irradiator). Eighteen weeks later, bone marrow cells were harvested fromrecipient mice and the presence of human cells was assessed inindividual mice by FACS using mouse anti-human CD45 (Inmmunotech,Villepinte-Roissy CDG, France), CD38, CD19, CD14-PE and CD34-PE-Cy5mAbs.

Human ALDP expression was studied by immunocytochemistry on at least 500bone marrow cells with an antibody that does not cross react with themouse ALDP (Fouquet et al., 1997).

Human CD34⁺ cells were purified from the bone marrow of two transplantedNOD/SCID mice and cultured in lympho-myeloid conditions (Sirven et al.,2000 and 2001).

Brain Immunohistochemistry

Deeply anesthetized animals were sacrificed. Brain was removed, frozeninto isopentane and stored at −80° C. until analysis. Serial sections(10 μm thick) were cut at −17° C. using a cryostat, fixed in 4%formaldehyde for 15 min and permreabilized in PBS-Triton X-100, 0.1%.Immunostaining of ALDP expressing cells and microglia was performed withanti-human ALDP antibody and Ricinus Conrnunis Agglutin (RCA) asdescribed (Fouquet et al., 1997). Cell nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI). Appropriate filters for each orcombined fluorochrome were used on a light microscope equipped forfluorescence (Nikkon E600).

In Situ Hybridization Histochemistry

Serial brain sections (10 μm thick) were cut at −17° C. using acryostat, fixed in 4% formaldehyde for 15 min and permeabilized inPBS-Triton X-100, 0.1%.

Non radioactive in situ hybridization was performed using a specifichuman Alu oligodeoxynucleotide probe labeled in 5′ with digoxigenin(Wilkinson, D.G. (ed). In Situ Hybridization. A practical Approach.Oxford University Press, New York, 1992).

After denaturation at 75° C. for 20 minutes, brain slides wereprehybridized in wet steamnroom chambers at 45° C. for 1.5 h. Slideswere then placed overnight at 45° C. in the hybridization solutioncontaining the probe (0.02 pmol/μl).

After 5 washes, antibody against digoxigenin conjugated to alkalinephosphatase (1:2000 dilution, Roche Diagnostics) was added anddigoxigenin was revealed with nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) (Promega).Labeled cells were counted on six sagittal brain sections of eachtransplanted mice.

Example 2 Transplantation of Human Modified CD34+ Cells canDifferentiate into Brain Microglia Expressing a Transgene. Results

Lentiviral Vector-Mediated ALD Gene Transfer into ALD Deficient CD34+Cells

A 36-hour long transduction protocol characterized by the absence ofcytokine prestimulation and low-cytokine serum-free medium was used.This allows effective transduction of CD34+ by lentiviral vectors in thelate G1 phase (Sutton, R. E. et al., J. Virol., 73, 3649-3660) but avoidterminal differentiation.

CD34+ cells from 3 ALD patients whose ALD gene mutation leaded to acomplete absence of ALD protein were used. After wash-out, cells wereincubated for 72 hours in long-term culture medium without cytokines.Transduction efficacy was then analysed by the expression of ALD proteinusing immunocytochemistry. 37.5 to 56.5% (mean 47.2%) of ALD cellsexpressed ALDP (Table 1).

To examine the transduction efficiency in colony-forming cells (CFCs),ALD deficient CD34+ cells were immediately plated on methylcelluloseafter transduction and cultured for 14-16 days. The number of individualCFC that expressed ALDP ranged from 32.5 to 39% (Table 1) (mean 36.6%).

No difference either in plating efficiency or CFU-GM/BFU ratio wasobserved in transduced and non-transduced cells (data not shown).

In vitro Analysis of Transduced ALD Deficient Hematopoietic Cells

In a perspective of CNS gene therapy, the main goal of gene transfer inhuman hematopoieitic stem cells is to target immature stem cells withproliferating and differentiating potentials in monocytes/macrophages inperipheral tissues and microglia in brain. To demonstrate that theTRIP-ΔU3-EF1α-ALD lentiviral vector was able to transduce ALD gene intosuch cells, different approaches were used.

First, transduced ALD deficient CD34+ cells were cultured in conditionsthat promote lympho-myeloid differentiation (Sirven et al., 2000 and2001). B, NK and myeloid cells were obtained. Monocytes-macrophages wereidentified with an anti-CD68 antibody and double immunostaining withanti-ALDP antibody. ALD CD34+ cells transduced with 35% efficacy (ALDpatient #2) could differentiate in CD68+ cells and 15% of these cellsexpressed ALDP.

Second, transduced CD34+ cells from 2 ALD patients (#1 and 2) weremaintained in long-term culture (LTC) for 5 weeks. ALDP was expressed in30% of 3000 cells that were studied at the end of the 2 LTC with novariation of this percentage between LTC experiments. Given thaterythroid precursors comprise 35% of all bone marrow cells and do notexpress ALDP, this allows to estimate that 46% of all LTC cellsexpressed ALDP after 5 weeks of culture.

LTC cells were then plated on methylcellulose and CFU-GM colonies wereindividually and randomly picked and scored for ALD expression. From atotal of 90 CFU-GM colonies (from the 3 LTC of patients #1 and #2), 44%expressed ALDP (Table 1), indicating that 44% of these transduced cellswere early hematopoietic progenitors.

To determine the transduction efficacy in LTC-IC cells (LTC-Ics), LTC oftransduced ALD CD34+ cells was performed in 96-well plates by platingone, ten or fifty CD34+ cells per well. After 5 weeks, cells from eachwell were plated on methylcellulose and the number of CFU-GM colonieswas scored after 15 days. The percentage of wells giving rise to CFU-GMcolonies is representative of the LTC-IC frequency of planted cells. 20%of transduced ALD deficient CD34+ cells were LTC-ICs, in agreement withthat observed with non transduced peripheral blood CD34+ cells.

ALDP expression was scored in CFU-GM colonies derived fromten-cell-wells. Colonies from each methylcellulose plate were pooled andanalysed. ALDP-expressing cells were found in every plate, meaning thatat least 50% of LTC-ICs have been transduced (20% of 10 transduced ALDCD34+ cells were LTC-IC, i.e. colonies obtained in each plate originatedfrom 2 cells. In each analysed plate, we found colonies expressing ALDP,meaning that at least one of the two LTC-IC from which they derivedexpressed ALDP). TABLE 1 Expression of ALD protein (ALDP) in human ALDdeficient CD34+ cells after lentiviral-mediated ALD gene transfer. % ofcells expressing % of individual % of individual ALD ALDP 72 hoursCFU-GM colonies CFU-GM colonies Patient after transduction expressingALDP derived from LTC #1  56.5 ± 13.1 38.5 ± 16.1 39; 43 (n = 4) (n = 3)(n = 2) #2 37.5 ± 3.5  39 ± 8.5 50 (n = 2) (n = 2) (n = 1) #3 47.5 ± 3.532.5 ± 3.5  ND (n = 2) (n = 2)*results are expressed as mean ± SD of n performed experiments.ND: not done.Functional Correction of ALD Biochemical Defect in Hematopoietic Cellsin vitro

ALD is biochemically characterized by the accumulation of VLCFAs thatinvolves mainly hexacosanoic (C_(26:0)) acid whereas the concentrationof docosanoic (C_(22:0)) acid remains normal. The C_(26:0)/C_(22:0)ratio thus reflects the ability of cells to metabolize VLCFA in thepresence of functional ALD protein (Dubois-Dalcq, M. et al., TrendsNeurosci., 22, 4-12, 1999).

Table 2 shows that the C_(26:0)/C_(22:0) ratio decreased proportionallyto the percentage of ALDP expression in ALD CD34+ cells, 72 hours aftertransduction, in CFU-GM derived cells and in transduced ALD CD34+cultured for 5 weeks (LTC). The correction of C_(26:0)/C_(22:0) ratiowas greater than expected with respect to the number of cells expressingALDP suggesting that overexpression of ALDP leads to increase VLCFAdegradation (Doerflinger et al., 1998). These results indicate thatlentiviral-vector encoded ALD protein was functional in peroxisomes oftransduced hematopoietic ALD cells. TABLE 2 Correction of very-longchain fatty acid (VLCFA) metabolism in ALD deficient CD34+ cells aftertransduction with a lentiviral vector and in derived CFU-GM colonies andLTC cells. % of transduced % of Observed C_(26:0)/C_(22:0) ALD cellsbiochemically ALD cells expressing corrected Control cellsNon-transduced Transduced ALDP ALD cells CD34+ cells 0.041 ± 0.0180.0192 ± 0.0421 0.118 ± 0.016 40 48 (n = 4) (n = 2) (n = 2) CFU-GM 0.042± 0.016 0.121 0.072 45 63 derived cells (n = 2) (n = 1) (n = 1) Cellsderived 0.021 ± 0.012 0.113 0.088 16.5 26 from 5-week (n = 2) (n = 1) (n= 1) LTCEngraftment of Transduced ALD Deficient CD34+ Cells in NOD/SCID Mice

Because long-term in vivo transplantatibility of human hematopoieticcells in NOD/SCID mice is considered a hallmark of cell immaturity (Dao,M. A. et al., Cur. Opin. Mol. Ther., 1, 553-557, 1999), we injected 10⁶to 1.5.10⁶ ALD CD34+ cells immediately after transduction into 5NOD/SCID mice.

Eighteen weeks after transplantation, mice were sacrificed and humanhematopoietic engraftment was analyzed by FACS of bone marrow cells withanti-human CD45 antibody. Two out of five NOD-SCID mice were engraftedwith transduced ALD deficient CD34+ cells in proportion ranging from 25%to 75% (Table 3). Human ALDP was expressed in 30 and 85% of bone marrowcells from recipient mice #3 and 8 respectively (Table 3).

The bone marrow cells of NOD/SCID mouse #3 engrafted with 75% CD45+human cells (Table 3; FIG. 1A) were phenotyped with specific humanantibodies against CD11b, CD14, CD15 and CD19 antibodies. 58% of humanCD45+ cells were B lymphocytes (CD19+) (FIG. 1B), 10% myeloid cells(CD15+) (FIG. 1B) and 1.75% monocytes (CD14+, CD 11+) (FIG. 1C).

Bone marrow from mouse #3 contained human CD34+/CD38− cells (FIG. 2),indicating that early human hematopoietic progenitor cells weremaintained in vivo.

Bone marrow CD34+ cells from mouse #3 were sorted by flow cytometry andcultured in conditions that promote lympho-myeloid differentiation(Sirven et al., 2000 and 2001). CD68 positive cells present in thisculture expressed ALDP indicating that long-term NOD/SCID repopulatingcells derived from transduced ALD deficient CD34+ cells were able todifferentiate into monocytes/macrophages and express recombinant ALDP inbone marrow.

Transduced Human Deficient ALD CD34+ Cells can Differentiate intoMicroglia and Express ALDP in the Brain of NOD/SCID Mice

In situ hybridization of brain slices from mouse #3 and #8 showed thepresence of human Alu positive cells in brain and cerebellum (FIGS. 3Aand 3B). 70±12 Alu positive cells per slice were present in the brainfrom mouse #3 and 5±2 in the brain slices from mouse #8 (Table 3).

Double immunostaining with antibodies against RCA (in green) and humanALDP (Cy3 in red) revealed the presence of human microglial cells thatexpressed ALDP in the brain both recipient NOD-SCID mice (FIG. 3C). 15±4ALDP positive cells per slice were present in the brain from mouse #3and 5±0.5 in the brain slices from mouse #8 (Table 3). These numbers areclose to what would be expected when taking into account the percentageof engraftment and human CD45+ ALDP positive cells in the bone marrowfrom these 2 NOD-SCID mice. This demonstrates that ALDP was expressed upto 4 months in human brain microglia present in the brain of NOD/SCIDmice that originate from transduced ALD deficient CD34+ cells.

Altogether, these data demonstrate that, in a model ofxeno-transplantation (the NOD-SCID mouse), human CD34+ can begenetically modified ex vivo to express a “therapeutic” protein and thatthese cells can differentiate in vivo into microglia and express in longterm (4 months) a genetically engineered “therapeutic” protein afterbone transplantation. TABLE 3 Analysis of ALDP positive cells in bonemarrow and brain from NOD-SCID mice % of human Expected Expected % ofCD45+ Number number number human cells of Alu of ALDP of ALDP CD45+expressing positive positive positive cells ALDP cells cells cellsNOD-SCID in bone in bone in brain in brain in brain Mouse marrow marrowper slice per slice per slice #3 75 30 70 ± 12 21 15 ± 4 #8 25 80 5 ± 2 4 4

Throughout this application, various publications are, referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

1-20. (canceled)
 21. A composition for the treatment of a subjectaffected by or susceptible to being affected by a CNS disorder, whereinthe composition comprises a population of human cells enriched in humancells that can express human CD34, wherein at least of portion of thecells comprises a nucleic acid of interest, and wherein the compositioncomprises the human cells in an amount sufficient to migrate to the CNSof a human subject and express the nucleic acid of interest in the CNSof the human subject when intravenously administered to the subject. 22.The composition according to claim 21, wherein cells in the compositionare capable of giving rise to microglia in the CNS when administered tothe human subject.
 23. The composition according to claim 21, whereinthe nucleic acid of interest encodes a polypeptide of interest, andwherein the composition comprises hematopoietic progenitor cells orhematopoietic stem cells isolated from cells obtained from the humansubject, and wherein the nucleic acid encoding the polypeptide ofinterest has been introduced into the isolated hematopoietic progenitorcells or hematopoietic stem cells under conditions that result in theexpression of the polypeptide of interest at a level that provides atherapeutic effect in the human subject.
 24. The composition accordingto claim 21, wherein the composition contains the human cells in anamount sufficient to reduce the severity of central nervous systemdamage or symptoms of a central nervous system disorder in the subject.25. The composition according to claim 23, wherein at least of portionof the cells are recombinant cells comprising the nucleotide sequenceencoding the polypeptide operably linked to expression control elements.26. The composition according to claim 25, wherein the compositioncontains the human cells in an amount sufficient to reduce the severityof central nervous system damage or symptoms of a central nervous systemdisorder in the subject.
 27. The composition according to claim 21,wherein the administered cells are hematopoietic progenitor orhematopoietic stem cells that can differentiate into microglia cells.28. The composition according to claim 21, wherein at least 20% of cellsin the composition express the CD34+ marker.
 29. The compositionaccording to claim 21, wherein the cells are isolated from the humansubject.
 30. The composition according to claim 22, wherein the cellsare recombinant cells comprising a nucleic acid of interest.
 31. Thecomposition according to claim 29, wherein at least a portion of thecells are cells that can express CD34 and are transduced with a vectorcomprising the nucleic acid of interest operably linked to a promotorcapable of effecting the expression of the nucleic acid of interest inthe cells.
 32. The composition according to claim 31, wherein at least aportion of the cells are transduced with a viral vector.
 33. Thecomposition according to claim 32, wherein the viral vector is alentiviral vector.
 34. The composition according to claim 23, whereinthe hematopoietic progenitor or hematopoietic stem cells express theCD34+ marker or are capable of differentiating into cells expressing theCD34+ marker.
 35. The composition according to claim 24, wherein thecells that can express human CD34 are hematopoietic progenitor cells orhematopoietic stem cells.
 36. The composition according to claim 21,wherein the nucleic acid of interest encodes a non-secreted or asecreted protein.
 37. A method of treating a subject affected by orsusceptible to being affected by a CNS disorder, wherein the methodcomprises administering to the subject a composition comprising apopulation of human cells enriched in human cells that can express humanCD34, wherein at least of portion of the cells comprises a nucleic acidof interest, and wherein the composition is administered to the subjectin an amount sufficient to migrate to the CNS of a human subject andexpress the nucleic acid of interest in the CNS of the human subjectwhen intravenously administered to the subject.
 38. The method asclaimed in claim 37, wherein the subject to be treated is pretreated inorder to enhance engraftment of the composition comprising the cells.39. The method as claimed in claim 37, wherein the CNS disorder, whichaffects or which is susceptible to affect the subject, is characterizedby diffuse neurodegeneration.
 40. The method as claimed in claim 37,wherein the CNS disorder is Alzheimer's disease.
 41. The method asclaimed in claim 37, wherein the administered cells are autologous tothe subject to be treated.
 42. The method as claimed in claim 37,wherein cells in the composition are capable of giving rise to microgliain the CNS when administered to the human subject.
 43. The method asclaimed in claim 37, wherein the nucleic acid of interest encodes apolypeptide of interest, and wherein the composition compriseshematopoietic progenitor cells or hematopoietic stem cells isolated fromcells obtained from the human subject, and wherein the nucleic acidencoding the polypeptide of interest has been introduced into theisolated hematopoietic progenitor cells or hematopoietic stem cellsunder conditions that result in the expression of the polypeptide ofinterest at a level that provides a therapeutic effect in the humansubject.
 44. The method as claimed in claim 27, wherein the compositioncontains the human cells in an amount sufficient to reduce the severityof central nervous system damage or symptoms of a central nervous systemdisorder in the subject.
 45. The method as claimed in claim 27, whereinat least a portion of the cells are recombinant cells comprising thenucleotide sequence encoding the polypeptide operably linked toexpression control elements.
 46. The method as claimed in claim 27,wherein the administered cells are hematopoietic progenitor orhematopoietic stem cells that can differentiate into microglia cells.47. The method as claimed in claim 27, wherein at least 20% of cells inthe composition express the CD34+ marker.
 48. The method as claimed inclaim 27, wherein the cells are isolated from the human subject.
 49. Themethod as claimed in claim 27, wherein at least a portion of the cellsare transduced with a viral vector.
 50. The method as claimed in claim49, wherein the viral vector is a lentiviral vector.
 51. The method asclaimed in claim 27, wherein the nucleic acid encodes a non-secreted ora secreted protein.